Method of screening candidate protective, potentiating, or inhibiting substances

ABSTRACT

The present invention relates to methods of treatment of programmed cell death (apoptosis) through the use of the HSV-1 gene γ 1 34.5 or the product of its expression, ICP34.5. The gene and its expression have been demonstrated to be.required for HSV-1 neurovirulence, and in particular, to act as an inhibitor of neuronal programmed cell death which allows for viral replication. Use of the gene therapy, or the protein itself, can be expected to result in inhibition of programmed cell death in various neurodegenerative diseases. This invention also relates to novel vectors for gene therapy, including modified herpes virus. Methods are presented for conducting assays for substances capable of mimicing, potentiating or inhibiting the expression of γ 1 34.5 or the activity of ICP34.5. Also, methods are disclosed for the treatment of tumorogenic diseases, including cancer, and for treatment of herpes and other viral infections using inhibitors of γ 1 34.5 expression or ICP34.5 activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of, commonly assigned application Ser. No. 08/419,853filed Apr. 11, 1995, now abandoned which is a division of applicationSer. No. 07/861,233 filed Mar. 31, 1992, now abandoned.

BACKGROUND OF THE INVENTION

The government may own certain rights in the present invention pursuantto grants from the National Cancer Institute (CA47451) and from theNational Institute for Allergy and Infectious Diseases (AI24009 andAI1588), and the United States Public Health Service.

1. Field of the Invention

The present invention is directed to methods for blocking or delayingprogrammed cell death, for delivery of gene therapy to specific cells,and for treatment of cancer and other tumorgenic diseases, as well astreatment of viral infections, through the potentiation of programmedcell death in tumor or viral host cells. The present invention is alsodirected to assays for candidate substances which can either inhibit, orpotentiate programmed cell death.

2. Description of the Related Art

a. Programmed Cell Death (Apoptosis)

In the last decade there has been increasing acceptance in thescientific community of the idea that cells may actually be internallyprogrammed to die at a certain point in their life cycle. As an activecellular mechanism programmed cell death, or apoptosis, has severalimportant implications. First, it is clear that such an active processcan provide additional means of regulating cell numbers as well as thebiological activities of cells. Secondly, mutations or cellular eventswhich potentiate apoptosis may result in premature cell death. Third, aform of cell death which is dependent on a specific active cellularmechanism can at least potentially be suppressed. Finally, an inhibitionof preprogrammed cell death would be expected to lead to aberrant cellsurvival and could be expected to contribute to oncogenesis.

In general, apoptosis involves distinctive morphological changesincluding nuclear condensation and degradation of DNA tooligonucleosomal fragments. In certain circumstances it is evident thatapoptosis is triggered by or is preceeded by changes in proteinsynthesis. Apoptosis appears to provide a very clean process forcellular destruction, in that the cells are disposed of by specificrecognition and phagocytosis prior to bursting. In this manner cells canbe removed from a tissue without causing damage to the surroundingcells. Thus, it can be seen that programmed cell death is crucial in anumber of physiological processes, including morphological development,clonal selection in the immune system, and normal cell maturation anddeath in other tissue and organ systems.

It has also been demonstrated that cells can undergo apoptosis inresponse to environmental information. Examples include the appearanceof a stimulus, such as glucocorticoid hormones for immature thymocytes,or the disappearance of a stimulus, such as interleukin-2 withdrawalfrom mature lymphocytes, or the removal of colony stimulating factorsfrom hemopoietic precursors (for a review of literature see Williams,Cell, 85; 1097-1098, Jun. 28, 1991). Furthermore, it has recently beendemonstrated that the response of removal to nerve growth factor fromestablished neuronal cell cultures mimics target removal, or axiotomy,or other methods of trophic factor removal, and it has been postulatedthat the cellular mechanism involved in this response is a triggering ofa suicide program or programmed cell death following the nerve growthfactor removal. (See Johnson et al., Neurobiol. of Aging, 10: 549-552,1989). The authors propose a “death cascade” or “death program”, whichenvisions that trophic factor deprivation initiates the transcription ofnew mRNA and the subsequent translation of that mRNA into deathassociated proteins which act in sequence to ultimately produce “killerproteins”. Such an intracellular mechanism seems to fit well with thecharacteristics of apoptosis discussed above, eg., death of specificcells without the release of harmful materials and without thedisruption of tissue integrity. Furthermore, the authors indicate thatinhibitors of macromolecular synthesis prevented the death of neurons inthe absence of nerve growth factor.

Studies have been conducted to explore the possibility that tumor cellscould be eliminated by artificially triggering apoptosis. The APO-1monoclonal antibody can induce apoptosis in several transformed human Band T cell lines. The antibody binds to a surface protein and could acteither by mimicking a positive death-inducing signal or by blocking theactivity of a factor required for survival. Also, anti-FAS antibodieshave similar effects, and the recent cloning and sequencing of the genefor the FAS antigen has shown that it is a 63 kilodalton transmembranereceptor. Itoh et al., Cell 66: 233-243 (1991).

However, it is important to note that neither APO-1 nor FAS can functionexclusively as triggers for cell death. Both are cell surface receptorsthat may activate quite different responses under other circumstances.Moreover, these antigens are not confined to tumor cells and theireffect on normal cells is certainly an important consideration, as isthe possible appearance of variants that no longer display the antigens.

It has also been demonstrated that the cell death induced by a range ofcytotoxic drugs, including several used in cancer therapy, has also beenfound to be a form of apoptosis. In fact, the failure of apoptosis intumor cells could be of fundamental importance in contributing not onlyto the evasion of physiological controls on cell numbers, but also toresistance both to natural defenses and to clinical therapy.

It has also been demonstrated that expression of the bcl-2 gene caninhibit death by apoptosis. The bcl-2 gene was isolated from thebreakpoint of the translocation between chromosomes 14 and 18 found in ahigh proportion of the most common human lymphomas, that beingfollicular B cell lymphomas. The translocation brings together the bcl-2gene and immunoglobulin heavy chain locus, resulting in an aberrantlyincreased bcl-2 expression in B cells. Subsequently, Henderson et al.(Cell, 65: 1107-1115, 1991) demonstrated that expression of latentmembrane protein 1 in cells infected by Epstein-Barr virus protected theinfected B cells from programmed cell death by inducing expression ofthe bcl-2 gene. Sentman et al. (Cell, 67: 879-88, Nov. 29, 1991)demonstrated that expression of the bcl-2 gene can inhibit multipleforms of apoptosis but not negative selection in thymocytes, andStrasser et al. (Cell, 67: 889-899, Nov. 29, 1991) demonstrated thatexpression of a bcl-2 transgene inhibits T cell death and can perturbthymic selfcensorship. Clem et al. (Science, 245: 1388-1390, Nov. 29,1991) identified a specific baculovirus gene product as beingresponsible for blocking apoptosis in insect cells.

b. Herpes Virus Infections and Neurovirulence

The family of herpes virus includes animal viruses of great clinicalinterest because they are the causative agents of many diseases.Epstein-Barr virus has been implicated in B cell lymphoma;cytomegalovirus presents the greatest infectious threat to AIDSpatients; and Varicella Zoster Virus, is of great concern in certainparts of the world where chicken pox and shingles are serious healthproblems. A worldwide increase in the incidence of sexually transmittedherpes simplex (HSV) infection has occurred in the past decade,accompanied by an increase in neonatal herpes. Contact with activeulcerative lesions or asymptomatically excreting patients can result intransmission of the infectious agent. Transmission is by exposure tovirus at mucosal surfaces and abraded skin, which permit the entry ofvirus and the initiation of viral replication in cells of the epidermisand dermis. In addition to clinically apparent lesions, latentinfections may persist, in particular in sensory nerve cells. Variousstimuli may cause reactivation of the HSV infection. Consequently, thisis a difficult infection to eradicate. This scourge has largely goneunchecked due to the inadequacies of treatment modalities.

The known herpes viruses appear to share four significant biologicalproperties:

1. All herpes viruses specify a large array of enzymes involved innucleic acid metabolism (e.g., thymidine kinase, thymidylate synthetase,dUTPase, ribonucleotide reductase, etc.), DNA synthesis (e.g., DNApolymerase helicase, primase), and, possibly, processing of proteins(e.g., protein kinase), although the exact array of enzymes may varysomewhat from one herpesvirus to another.

2. Both the synthesis of viral DNAs and the assembly of capsids occur inthe nucleus. In the case of some herpes viruses, it has been claimedthat the virus may be de-enveloped and re-enveloped as it transitsthrough the cytoplasm. Irrespective of the merits of these conclusions,envelopment of the capsids as it transits through the nuclear membraneis obligatory.

3. Production of infectious progeny virus is invariably accompanied bythe irreversible destruction of the infected cell.

4. All herpes viruses examined to date are able to remain latent intheir natural hosts. In cells harboring latent virus, viral genomes takethe form of closed circular molecules, and only a small subset of viralgenes is expressed.

Herpes viruses also vary greatly in their biologic properties. Some havea wide host-cell range, multiply efficiently, and rapidly destroy thecells that they infect (e.g., HSV-1, HSV-2, etc.). Others (e.g., EBV,HHV6) have a narrow host-cell range. The multiplication of some herpesviruses (e.g., HCMV) appears to be slow. While all herpes viruses remainlatent in a specific set of cells, the exact cell in which they remainlatent varies from one virus to another. For example, whereas latent HSVis recovered from sensory neurons, latent EBV is recovered from Blymphocytes. Herpes viruses differ with respect to the clinicalmanifestations of diseases they cause.

Herpes simplex viruses 1 and 2 (HSV-1, HSV-2), are among the most commoninfectious agents encountered by humans (Corey and Spear, N. Ena. J.Med., 314: 686-691, 1986). These viruses cause a broad spectrum ofdiseases which range from mild and nuisance infections such as recurrentherpes simplex labialis, to severe and life-threatening diseases such asherpes simplex encephalitis (HSE) of older children and adults, or thedisseminated infections of neonates. Clinical outcome of herpesinfections is dependent upon early diagnosis and prompt initiation ofantiviral therapy. However, despite some successful therapy, dermal andepidermal lesions recur, and HSV infections of neonates and infectionsof the brain are associated with high morbidity and mortality. Earlierdiagnosis than is currently possible would improve therapeutic success.In addition, improved treatments are desperately needed.

Extrinsic assistance has been provided to infected individuals, inparticular, in the form of chemicals. For example, chemical inhibitionof herpes viral replication has been effected by a variety of nucleosideanalogues such as acyclovir, 5-flurodeoxyuridine (FUDR),5-iododeoxyuridine, thymine arabinoside, and the like.

Some protection has been provided in experimental animal models bypolyspecific or monospecific anti-HSV antibodies, HSV-primedlymphocytes, and cloned T cells to specific viral antigens (Corey andSpear, N. Eng. J. Med., 314: 686-691, 1986). However, no satisfactorytreatment has been found.

The γ₁34.5 gene of herpes simplex virus maps in the inverted repeatregion of the genome flanking the L component of the virus. Thediscovery and characterization of the gene was reported in severalarticles (Chou and Roizman, J. Virol., 57: 629-635, 1986, and J. Virol.,64: 1014-1020, 1990; Ackermann et al., J. Virol., 58: 843-850, 1986).The key features are: (i) the gene encodes a protein of 263 amino acidin length; (ii) the protein contains Ala-Thr-Pro trimer repeat ten timesin the middle of the coding sequence; (iii) the protein is basic innature and consists of large number of Arg and Pro amino acids; (iv) thepromoter of the gene maps in the a sequence of the genome which alsoserves several essential viral functions for the virus; (v) thecis-acting element essential for the expression of the gene γ₁34.5 iscontained within the a sequence, in particular, the DR2 (12 base pairsequence repeated 22 times) and U_(b) element. This type of promoterstructure is unique to this gene and not shared by other viral genepromoters.

The function of the gene γ₁34.5 in its ability to enable the virus toreplicate, multiply and spread in the central nervous system (CNS) wasdemonstrated by a set of recombinant viruses and by testing theirabilities to cause fatal encephalitis in the mouse brain. The mutantviruses lacking the gene therefore lost their ability to multiply andspread in the CNS and eyes and therefore is non-pathogenic. See Chou etal., Science, 250: 1212-1266, 1990.

The γ₁34.5 gene functions by protecting the nerve cells from totalprotein synthesis shutoff in a manner characteristic of programmed celldeath (apoptosis) in neuronal cells. The promoter appears to containstress response elements and is transactivated by exposure to UVirradiation, viral infection, and growth factor deprivation. These datasuggest that the gene γ₁34.5 is transactivated in the nerve cells attimes of stress to prevent apoptosis.

The significance of these findings therefore lies in the fact thatγ₁34.5 extends viability or lends protection to the nerve cells so thatin this instance, the virus can replicate and spread from cell tocell—defined as neurovirulence. It also appears that the protection canbe extended to other toxic agents or environmental stresses to which thecell is subjected. An important aspect about the nature of the neurons,unlike any other cells in human, is the fact that neurons in the brain,eyes or CNS do not regenerate which forms the basis of many impairedneurological diseases. Any genes or drugs that extend the life of cellsfrom death or degeneration can be expected to have a significant impactin the area of neural degeneration.

The role of γ₁34.5, and anti-apoptosis factors, in infected cells is inits early stages of elucidation. Recent studies have suggested thatEpstein-Barr virus enhances the survival capacity of infected cellsthrough latent membrane protein 1(LMP1)-induced up-regulation of bcl-2.In that system it is postulated that LMP 1 induced bcl-2 up regulationgives virus infected B cells the potential to by-pass physiologicalselection and gain direct access to long lived memory B cell pools.However, bcl-2 expression fails to suppress apoptosis in somesituations, for example upon withdrawal of interleukin-2 orinterleukin-6. Moreover, the intracellular mechanism of action of bcl-2expression remains unknown.

c. Programmed Cell Death and Disease Therapy

In light of the foregoing, it is apparent that the expression of γ₁34.5in CNS cells added an extra dimension of protection to the neuronsagainst viral infection, and naturally ocurring and stress-inducedapoptosis. An appreciation of this extra dimension of protection can beutilized in novel and innovative means for control and treatment ofcentral nervous system (CNS) disorders. Specifically, treatment of CNSdegenerative diseases, including Alzheimer's disease, Parkinson'sdisease, Lou Gerig's disease, and others the etiology of which may betraceable to a form of apoptosis, and the treatment of which iscurrently very poor, could be improved significantly through the use ofeither the γ₁134.5 gene in gene therapy or the protein expressed byγ₁34.5 as a therapeutic agent. This is especially critical where thedeath of neuronal cells is involved, due to the fact that, as noted,such cells do not reproduce post-mitotically. Since a finite number ofneurons are available it is crucial to have available methods and agentsfor their protection and maintenance. γ₁134.5 is also a very useful genefor assays of substances which mimic the effect of γ₁34.5and blockstress of biologically induced programmed cell death.

Furthermore, the HSV-1 virus, appropriately modified so as to be madenon-pathogenic, can serve as a vehicle for delivery of gene therapy toneurons. The HSV-1 virus is present in neurons of the sensory ganglia of90% of the world's human population. The virus ascends into neuronalcell bodies via retrograde axonal transport, reaching the axon from thesite of infection by the process of neurotropism. Once in the neuronalcell body the virus remains dormant until some form of stress inducesviral replication (e.g. UV exposure, infection by a second virus,surgery or axotomy). As noted, the use of HSV-1 as a vector wouldnecessitate construction of deletion mutants to serve as safe,non-pathogenic vectors. Such a virus would act as an excellent vectorfor neuronal gene therapy and its use would be an especially importantdevelopment since few methods of gene therapy provide a means fordelivery of a gene across the central nervous system's blood-brainbarrier.

Moreover, other viruses, such as HSV-2, picornavirus, coronavirus,eunyavirus, togavirus, rahbdovirus, retrovirus or vaccinia virus, areavailable as vectors for γ₁34.5 gene therapy. As discussed with regardto the use of HSV-1 viruses, these vectors would also be altered in sucha way as to render them non-pathogenic. In addition to the use of anappropriately mutated virus, implantation of transfected multipotentneural cell lines may also provide a means for delivery of the γ₁134.5gene to the CNS which avoids the blood brain barrier.

In addition, use of the HSV-1 virus with a specific mutation in theγ₁34.5 gene provides a method of therapeutic treatment of tumorogenicdiseases both in the CNS and in all other parts of the body. The “γ₁34.5minus” virus can induce apoptosis and thereby cause the death of thehost cell, but this virus cannot replicate and spread. Therefore, giventhe ability to target tumors within the CNS, the γ₁34.5 minus virus hasproven a powerful therapeutic agent for hitherto virtually untreatableforms of CNS cancer. Furthermore, use of substances, other than a virus,which inhibit or block expression of genes with antiapoptotic effects intarget tumor cells can also serve as a significant development in tumortherapy and in the treatment of herpes virus infection, as well astreatment of infection by other viruses whose neurovirulence isdependent upon an interference with the host cells' programmed celldeath mechanisms.

SUMMARY OF THE INVENTION

This invention relates to methods for the prevention or treatment ofprogrammed cell death, or apoptosis, in neuronal cells for therapy inconnection with neurodegenerative diseases, as well as methods oftreatment of cancer and other tumorogenic diseases and herpes virusinfection. The present invention also relates to assay methodologiesallowing for the identification of substances capable of modulating theeffects of the γ₁34.5 gene or its protein expression product ICP34.5,i.e., substances capable of potentiating or inhibiting their effects.Additionally, the present invention also relates to assay methodologiesdesigned to identify candidate substances able to mimic either γ₁34.5expression or the activity of ICP34.5. The present invention alsorelates to methods of delivering genes to cells for gene therapy.

In one illustrative embodiment of the present invention a method ofpreventing or treating programmed cell death in neuronal cells isdescribed in which a non-pathogenic vector is prepared which containsthe γ₁34.5 gene. This vector is then introduced into neuronal cellswhich are presently undergoing or are likely to undergo programmed celldeath. Those skilled in the art will realize that several vectors aresuitable for use in this method, although the present inventionenvisions the use of certain unique and novel vectors designedspecifically for use in connection with delivery of the γ₁34.5 gene.

One such vector envisioned by the present invention is the HSV-1 virusitself, modified so as to render it non-pathogenic. Because of theunique capability of the HSV-1 virus to use an axon's internal transportsystem to move from the peripheral nerve endings of the neuron into theneuronal cell body, the present invention proposes the use of thenon-pathogenic HSV-1 virus injected into the vicinity of the synapticterminals of affected neurons, or in the area of a peripheral wound orlesion or other appropriate peripheral locus. The HSV-1 virus containingthe γ₁34.5 gene, under a different target-specific promoter, would thenbe transported into the neuronal cell body via retrograde axonaltransport.

The present invention envisions specific genomic modifications beingintroduced into the HSV-1 virus in order to render the virusnon-cytotoxic. These modifications could include deletions from thegenome, rearrangements of specific genomic sequences, or other specificmutations. One example of such a modification comprises modification ordeletion of the α4 gene which encodes the ICP4 protein. Deletion ormodification of the gene expressing ICP4 renders the HSV-1 virus unableto express genes required for viral DNA and structural proteinsynthesis. However, the γ₁34.5 gene placed under a suitable promoterwould be expressed, thus inducing an anti-apoptotic effect in the neuronwithout the potential for stress induced neurovirulence. Other geneswhich might be modified include the the α0 gene. The present inventionalso envisions the use of other vectors including, for example,retrovirus, picorna virus, vaccinia virus, HSV-2, coronavirus,eunyavirus, togavirus or rhabdovirus vectors. Again, use of of suchviruses as vectors will necessitate construction of deletion mutationsso that the vectors will be safe and non-pathogenic.

Another method by which the present invention envisions introducing theγ₁34.5 gene into neuronal cells undergoing or likely to undergoprogrammed cell death, is through the use of multi-potent neural celllines. Such lines have been shown to change phenotype in vitro and havealso been demonstrated to become integrated into the central nervoussystem of mice and to differentiate into neurons or glia in a mannerappropriate to their site of engraftment. Snyder, et al., Cell, 68:33-51, 1992. Transplant or engraftment of multi-potent neural cell linesinto which the γ₁34.5 gene has been introduced into an area of thecentral nervous system in which cells are undergoing or are likely toundergo programmed cells death is expected to lead to reversal andinhibition of programmed cell death.

It is expected that the ability of γ₁34.5 to inhibit apoptosis will be aboon not only in human medicine, but also in basic scientific research.In this regard the present invention also envisions the use of theγ₁34.5 gene in the extension of the life of neuronal cells in cellculture. Introduction of a non-cytotoxic vector into cultured neuronalcells will have an anti-apoptotic effect and will thereby extend thelife of cell cultures. This in turn will extend the time periods overwhich experimentation may be conducted, and can also be expected todecrease the cost of conducting basic research.

In addition to utilizing a vector comprising the γ₁34.5 gene, thepresent invention also discloses a method of preventing or treatingprogrammed cell death in neuronal cells which involves the use of theproduct of expression of the γ₁34.5 gene. The protein expressed byγ₁34.5 is called ICP34.5. Ackermann, et al. (J. Virol., 58: 843-850,1986) reported that ICP34.5 has an apparent molecular weight of 43,500upon SDS-polyacrylamide gel electrophoresis, appears to accumulatelargely in the cytoplasm of HIV infected cells, and in contrast to manyHSV-1 proteins, ICP34.5 has been demonstrated to be soluble inphysiologic solutions.

In practicing this method or the method in which the γ₁34.5 gene isintroduced into cells, it is envisioned that the γ₁34.5 gene or abiological functional equivalent thereof could be used for gene therapy,or ICP34.5 in a purified form or a biological functional equivalent ofthe ICP34.5 protein could be utilized as an anti-apoptotic agent. Asused herein, functional equivalents are intended to refer to thoseproteins, and their encoding nucleic acid sequences, in which certainstructural changes have been made but which nonetheless are, or encode,proteins evidencing an effect similar to that of ICP34.5.

In light of the fact that certain amino acids may be substituted forother amino acids in a protein without appreciable loss of definedfunctional activity, it is contemplated by the inventors that variouschanges may be made in the sequence of the ICP34.5 protein (or theunderlying DNA of the γ₁34.5 gene) without an appreciable loss ofbiological utility or activity. Amino acids with similar hydropathicscores may be substituted for one another (see Kyte et al., J. Mol.Biol., 157: 105-132, 1982, incorporated herein by reference), as mayamino acids with similar hydrophilicity values, as described in U.S.Pat. No. 4,554,101, incorporated herein by reference.

Therefore, amino acid substitutions are generally based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions which take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include:arginine and lysine; glutamate and aspartate; serine and threonine;glutamine and asparagine; and valine, leucine and isoleucine.

This embodiment of the present invention describes a method whichinvolves combining ICP34.5 or a biological functional equivalent thereofwith a pharmaceutically acceptable carrier in order to form apharmaceutical composition. (It should be understood in subsequentdiscussions that when γ₁34.5 or ICP34.5 are referred to, the inventorsintend to include biological functional equivalents, including anychemicals which mimic the effect of γ₁34.5.) Such a composition wouldthen be administered to neurons likely to undergo or undergoingprogrammed cell death. Such a composition could be administered to ananimal using intravenous, intraspinal injection or, in certaincircumstances, oral, intracerebral or intraventricular administrationmay be appropriate. Furthermore, neuronal cells in culture could alsobenefit from administration of ICP34.5 through administration directlyinto the medium in which the neuronal cells are grown.

ICP34.5 can be prepared using a nucleic acid segment which is capable ofencoding ICP34.5 (i.e., the γ₁34.5 gene or a biological functionalequivalent). Such a segment could be expressed using, for example, atechnique involving transferring the γ₁34.5 segment into a host cell,culturing the host cell under conditions suitable for expression of thesegment, allowing expression to occur, and thereafter isolating andpurifying the protein using well established protein purificationtechniques. The nucleic acid segment would be transferred into hostcells by transfection or by transformation of a recombinant vector intothe host cell.

A particularly important embodiment of the present invention relates toassays for candidate substances which can either mimic the effects ofthe γ₁34.5 gene, or mimic the effects of ICP34.5, as well as assays forcandidate substances able to potentiate the function of γ₁34.5 orpotentiate the protective function of ICP34.5. Additionally, methods forassaying for candidate substances able to inhibit either γ₁34.5expression or the activity of ICP34.5 are also embodiments of thepresent invention.

In an exemplary embodiment, an assay testing for candidate substanceswhich would block the expression of the anti-apoptosis gene or inhibitthe activity of an anti-apoptotic protein such as ICP34.5 would proceedalong the following lines. A test plasmid construct bearing the asequence promoter and portions of the coding sequence of γ₁34.5 is fusedto the lacZ reporter gene, or any other readily assayable reporter gene.This construct is then introduced into an appropriate cell line, forexample a neuroblastoma or PC12 cell line, by G418 selection. A clonaland continuous cell line for screening purposes is then established. Acontrol plasma construct bearing an HSV late promoter (a promoter whichwould normally not be expressed in cell lines and not induced to expressby a stress factor which would normally induce apoptosis) is fused tothe same or different indicator gene. This construct is also introducedinto a continuous clonal cell line and serves as a control for the testcell line. The anti-apoptosis drugs would then be applied. Environmentalstresses which typically trigger a sequence promoter activation andcause programmed cell death, such as UV injury, viral infection ordeprivation of nerve growth factor, would then be applied to the cells.In control cells, the stress should have no effect on the cells andproduce no detectible reaction in the assay. Stress in a test cell linein the absence of a positive candidate substance would give rise to anappropriate reaction, typically a colorimetric reaction. Introduction ofstress to the test cell line in the presence of the candidate substancewould give rise to an opposite calorimetric reaction indicating that thecandidate substance interferes either with expression of the γ₁34.5gene, or with the ability of the substance to interfere with theanti-apoptotic activity of ICP34.5.

Similarly, the present invention describes an assay for candidatesubstances which would mimic or potentiate the activity of ICP34.5, orwhich would mimic the expression of γ₁34.5, and such an assay wouldproceed along lines similar to those described above. A test cell line(e.g., a neuroblastoma cell line) constitutively expressing ICP34.5 anda fluorescent tagged cellular gene or any other tag providing an easilydetected marker signalling viability of the cells is produced. Inaddition, a corresponding null cell line consisting of an appropriateindicator gene, for example the a-lacZ indicator gene, and the same hostindicator gene as in the test cell line is also produced. Also, a thirdcell line (e.g., a vero cell line) consisting of the same indicator geneand the identical host indicator gene is also produced. Again,environmental stresses which trigger programmed cell death in theabsence of γ₁34.5 are applied to the cells. Candidate substances arealso applied in order to determine whether they are able to mimic orpotentiate the anti-apoptotic effects of γ₁34.5 expression or theanti-apoptotic activity of ICP34.5 or biological functional equivalentsthereof.

The present invention also embodies a method of delivering genes forgene therapy. In an exemplary embodiment, the method involves combiningthe gene used for gene therapy with a mutated virus such as thosedescribed above, or with the HSV-1 virus rendered non-pathogenic. Thegene and the virus are then combined with a pharmacologically acceptablecarrier in order to form a pharmaceutical composition. Thispharmaceutical composition is then administered in such a way that themutated virus containing the gene for therapy, or the HSV-1 wild typevirus containing the gene, can be incorporated into cells at anappropriate area. For example, when using the HSV-1 virus, thecomposition could be administered in an area where synaptic terminalsare located so that the virus can be taken up into the terminals andtransported in a retrograde manner up the axon into the axonal cellbodies via retrograde axonal transport. Clearly, such a method wouldonly be appropriate when cells in the peripheral or central nervoussystem were the target of the gene therapy.

The present invention also envisions methods and compositions for thetreatment of cancer and other tumorogenic diseases, as well as herpesinfections or other infections involving viruses whose virulence isdependent upon an anti-apoptotic effect. Candidate substances identifiedas having an inhibiting effect upon either the expression or activity ofICP34.5 identified in the assay methods discussed above could be used toinduce cell death in target tumor cells, or in virus-infected cells.Pharmaceutical compositions containing such substances can be introducedusing intrathecal, intravenous, or direct injection into the tumor orthe infected area, as appropriate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth the relationship between FIGS. 1 a-1 d and how thesefigures should be compiled and viewed for a proper understanding oftheir content.

FIGS. 1 a-1 d show DNA sequence comparisons of HSV-1 strains F, 17syn+,MGH-10, and CVG-2 in the region of the gene for ICP34.5 (FIGS. 1 a and 1b) and the predicted open frames for ICP34.5 in these strains (FIGS. 1 cand 1 d). Unless otherwise indicated by a new base (insertion of A, C,G, or T), a new amino acid (three-letter code), or absence of a base oramino acid (−), the sequences for strains HSV-1(17)syn+, HSV-1(MGH-10),and HSV-1(CVG-2) were identical to the sequence for HSV-1(F). Anasterisk indicates initiation of a repeat sequence of nine nucleotidesor three amino acids. Direct repeat 1(DR1) designates the 20-base-pairrepeat sequence flanking the a sequence. Sequences upstream of directrepeat 1 are contained within the a sequence. The number at the end ofeach line indicates the relative position from nucleotide 1 (FIGS. 1 aand 1 b) or amino acid 1 (FIGS. 1 c and 1 d). The initiation andtermination codons for the HSV-1(F) sequence are underlined.

FIG. 2 shows sequence arrangements of the genome of wild-type strainHSV-1 strain F [HSV-1(F)] and of recombinant viruses derived from it.Top line, the sequence arrangement of HSV-1(F) Δ305. The rectanglesidentify the inverted repeats ab, b′ a′ c, and ca. The HSV-(F) asequence is present in a direct orientation at the two genomic terminiand in the inverted orientation at the junction between the long andshort components. The b and c sequences are approximately 9 and 6 kbplong, respectively. The triangle marked TK identifies the position ofthe tk gene and of the Bg1 II to Sac I sequence of BamHI Q fragmentdeleted from HSV-1(F)Δ305. Lines two and three from the top show thatthe b sequences contain the genes specifying ICP34.5 and ICPO and, sinceb sequence is repeated in an inverted orientation, there are two copiesof these genes per genome. The construction of the a24-tk fragementcontaining portions of the glycoprotein H gene has been described. Chouand Roizman, J. Virol., 57: (1986); Ackerman et al., J. Virol., 58: 843(1986); Chou and Roizman, J. Virol., 64: 1014 (1990). Line 7 shows aschematic diagram of the insertion of the oligonucleotide containingstop codons in all three reading frames. The plasmids pRB3615 andpRB2976 used in the construction of R4002 and R4004, respectively, weredescribed elsewhere. Chou and Roizman, J. Virol., 57: 629 (1986) and J.Virol., 64: 1014 (1990). To generate pRB3616, plasmid pRB143 wasdigested with BstEII and Stu I, blunt-ended with T4 polymerase, andrelegated. The asterisks designate nucleotides from vector plasmid thatform cohesive ends with the synthesized oligomers. The insertion of theα4 epitope into the first amino acid of ICP34.5 (line 9) has beendescribed, Chou and Roizman, J. Virol., 64: 1014 (1990), except that inthis instance the sequence was inserted into both copies of the γ₁34.5gene. The tk gene was restored in all recombinant viruses tested inmice. HSV-1(F)R (line 6) was derived from R3617 by restoration of thesequences deleted in γ₁34.5 and tk genes. N, Be, S, and St areabbreviations for Nco I, BstEII, Sac I, and Stu I restrictionendonucleases (New England Biolabs), respectively. The numbers inparentheses are the tk⁺ version of each construct tested in mice.

FIGS. 3 a and 3 b show an autoradiographic image of electrophoreticallyseparated digest of plasmid, wild-type, and mutant virus DNAs,transferred to a solid substrate and hybridized with labelled probes forthe presence of γ₁34.5 and tk genes. The plasmids or viral DNAs shownwere digested with BamHI or, in the case of R4009 shown in lanes 10,with both BamHI and Spe I. The hybridization probes were the fragmentNco I to Sph I contained entirely within the coding sequences of γ₁34.5(FIG. 3 a) and the BamHI Q fragment of HSV-1(F) (FIG. 3 b). The probeswere labeled by nick translation of the entire plasmid DNAs with [α-³²P]deoxycytidine triphosphate and reagents provided in a kit (Du PontBiotechnology Systems). The DNAs that were limit digested with BamHI(all lanes) or both BamHI and Spe I (left panel, lane 10) wereelectrophoretically separated on 0.8% agarose gels in 90 mMtrisphosphate buffer at 40 V overnight. The DNA was then transferred bygravity to two nitrocellulose sheets sandwiching the gel and hybridizedovernight with the respective probes. γ₁34.5 maps in BamHI S and SPfragments, which form a characteristic ladder of bands at 500-bpincrements. The ladders are a consequence of a variable number of asequences in the repeats flanking the unique sequences of the junctionbetween the long and short components, whereas BamHI S is the terminalfragment of the viral genome at the terminus of the long component,whereas BamHI SP is a fragment formed by the fusion of the terminalBamHI S fragment with BamHI P, the terminal BamHI fragment of the shortcomponent. Bands of BamHI S, SP, and Q and their deleted versions,ABamHI S, ABamHI SP, and ABamHI Q (AQ), respectively, are indicated.Band 1 represents the 1.7-kbp α27-tk insert into the BamHI SP fragmentin R4002, and therefor this fragment reacted with both labeled probes(lanes 4). Band 2 represents the same insertion into the BamHI Sfragment.

FIGS. 4 a and 4 b show autoradiographic images (FIG. 4 a) and photographof lysates of cells mock infected (M) or infected with HSV-1(F) andrecombinant viruses (FIG. 4 b) separated electrophoretically indenaturing polyacrylamide (10%) gels, transferred electrically to anitrocellulose sheet, and stained with rabbit polyclonal antibody R4described elsewhere. Ackerman et al., J. Virol., 58: 843 (1986); Chouand Roizman, J. Virol., 64: 1014 (1990). Replicate cultures of Verocells were infected and labeled with [³⁵S]methionine (Du PontBiotechnology Systems) from 12 to 24 hours after infection, andequivalent amounts of cell lysates were loaded in each slot. Theprocedures were as described (Ackerman et al.; Chou and Roizman) exceptthat the bound antibody was made apparent with the alkaline phosphatasesubstrate system supplied by Promega, Inc. Infected cell proteins weredesignated by number according to Honess and Roizman (J. Virol., 12:1346 (1973)). The chimeric ICP34.5specified by R4003 migrated moreslowly than the protein produced by other viruses because of theincreased molecular weight caused by the insertion of the epitope.

FIG. 5 is a schematic representation of the genome structure andsequence arrangements of the HSV-1 strain F [HSV01(F)] and relatedmutants. Top line: The two covalently linked components of HSV-1 DNA, Land S, each consist of unique sequences flanked by inverted repeats (7,31). The reiterated sequences flanking the L component designated as abad b′a′ are each 9 kb in size, whereas the repeats flanking the Scomponent are 6.3 kb in size (31). Line 2: expansion of portions of theinverted repeat sequences ab and b′a′ containing the γ₁34.5 and α0genes. Line 3: sequence arrangement and restriction endonuclease sitesin the expanded portions shown in line 2. Open box represents the 20 bpdirect repeat sequence (DR1), flanking the a sequence (26,27).Restriction site designations are N,-NcoI; Be,-BstEII; S,SacI; St,-StuI.Line 4: the thin line and filled rectangle represent the transcribed andcoding domains of the γ₁34.5 gene (406). Vertical line, location of thetranscription initiation sites of γ₁34.5 and of α0 genes. In the R3616viral recombinant, one Kb was deleted between BstEII at 28th amino acidof γ₁34.5 to StuI at the 3′ terminus of the genes as shown. In HSV-1(F)RDNA, the sequences deleted from the γ₁34.5 gene in R3616 were restoredand therefore the virus could be expected to exhibit a wild-typephenotype. The R4009 recombinant virus DNA contains an in frametranslation termination codons at the BstEII site. Vertical arrow on toppoints to the site of the stop codon insertion.

FIGS. 6 a and 6 b shows an autoradiographic image of eletrophoreticallyseparated lysates of infected cells labeled for 90 minutes with³⁵S-methionine at stated time points. The SK-N-SH neuroblastoma and Verocell lines were mock infected (M) or exposed at 37° C. to 5 pfu ofwild-type or mutant viruses in 6 well (Costar, Cambridge, Mass.) dishes.At 2 hours post exposure, the cells were overlaid with mixture 199supplemented with 1% calf serum. At 5.5 and 11.5 hours post exposure ofcells to viruses, replicate infected 6 well cultures were overlaid with1 ml of the 199v medium lacking unlabeled methionine but supplementedwith 50 μCi of ³⁵S-methionine (specific activity >1,000 Ci/mmole,Amersham Co. Downers Grove, Ill.). After 90 minutes in labeling medium,the cells were harvested, solubilized in a buffer containing sodiumdodecyl sulphate, subjected to electrophoresis on a denaturing 12%polyacrylamide gels crosslinked with N, N′ Diallytartardiamide,electrically transferred to nitrocellulose sheet and subjected toautoradiography as previously described (13). Infected cell polypeptides(ICP) were designated according to Honess and Roizman, J. Virol., 12:1347-1365 (1973).

FIGS. 7 a and 7 c show autoradiographic images of labeled polypeptideselectrophoretically separated in denaturing gels and photographs ofprotein bands made apparent by their reactivity with antibodies. TheSK-N-SH neuroblastoma and Vero cells were either mock infected (M) orinfected with 5 pfu of either R3616 or the parent HSV-1(F) per cell asdescribed in the legend to FIGS. 6 a and 6 b. The cultures were labeledfor 1.5 hr before harvesting at 13th hr post exposure of cells to virus.Preparation of cell extracts, electrophoresis of the polypeptides,electric transfer of the separated polypeptides to a nitrocellulosesheet, and autoradiography were carried out as described elsewhere(Ackerman et al., J. Virol., 52: 108-118, 1984). The nitrocellulosesheets were reacted with the respective antibodies with the aid of kitsfrom Promega, Inc. (Madison, Wis.) according to manufacture'sinstruction. Monoclonal antibodies H1142 against α27 and H725 againstthe product of the U_(L)26.5 gene were the generous gift of LenorePereira, University of California at San Francisco. The M28 monoclonalantibody to U_(S)11 protein and the rabbit polyclonal antibody R161against viral thymidine kinase (βtk) were made to a specific peptide (M.Sarmiento and B. Roizman, unpublished studies) in this laboratory. ICPdesignations were the same as noted before.

FIG. 8 shows an autoradiographic image of viral proteins expressedduring infection on SK-N-SH neuroblastoma cell lines in the presence orabsence of phosphonoacetate (PAA). Duplicate SK-N-SH neuroblastoma cellcultures were either treated with phosphonoacetate (300 μg/ml; SigmaChemical Co., St. Louis, Mo.) starting at 1.5 hr prior to infectioncontinuously until the termination of infection or left untreated. Thecultures were either mock infected or exposed to 5 pfu of eitherHSV-1(F), R3616, R4009 and HSV-1(F)R at 5 pfu/cell. At 11.5 hours postexposure to virus, the cells were overlaid with medium containing 50 μCiof ³⁵S-methionine as described in legend to FIG. 6. Polypeptideextraction, electrophoresis on 12% polyacrylamide gels crosslinked withN,N′ Diallytartardiamide, electrical transfer to nitrocellulose sheetsand autoradiography were as described in the legend to FIGS. 6 a and 6b.

FIGS. 9 a and 9 b show viral DNA and RNA accumulation in infectedSK-N-SH neuroblastoma and Vero cell cultures. FIG. 9 a: Photograph ofethidium bromide stained agarose gel containing electrophoreticallyseparated BamHI digests of total DNAs extracted from mock-infected cellsor cells infected with HSV-1(F), R3616, R4009 or HSV-1(F)R viruses. FIG.9 b: Hybridization of electrophoretically separated RNA transferred to anitrocellulose sheets probed with RNA sequences antisense to α47,U_(S)10 and U_(S)11 open reading frames. SK-N-SH neuroblastoma and Verocells were either mock-infected or exposed to 5 pfu of HSV1(F) or ofR3616 per cell. Total DNAs were extracted from cells at 17 hr postinfection by the procedure published by Katz et al., (J. Virol., 64:4288-4295), digested with BamHI, electrophoresed in 0.8% agarose gel at40V overnight and stained with ethidium bromide for visualization. ForRNA analysis, SK-N-SH neuroblastoma and Vero cells were eithermock-infected or infected with R3616 and HSV-1(F) as described above. At13 hrs post exposure of cells to virus the RNA was extracted by theprocedure of Peppel and Baglioni (BioTechniques, 9: 711-712, 1990). TheRNAs were then separated by electrophoresis on 1.2% agarose gel,transferred by gravity to a nitrocellulose sheet and probed withanti-sense RNA made from in vitro transcription of pRB3910 off T7promoter using kit from Promega, Inc. according to manufacturer'sinstruction. α47, U_(S)10 and U_(S)11 transcripts overlap in sequenceand share the same 3′ co-terminal sequence. McGeoch et al., J. Gen.Virol., 64: 1531-1574 (1988). The U_(S)10 transcript is of low abundanceand not detected in this assay.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Introduction

The present invention relates to the use of the HSV-1 γ₁34.5 gene, theICP34.5 protein expressed by that gene, and derivatives of the proteinwhich function in a similar manner as therapeutics for (neuronal)programmed cell death. The present invention also relates to the use ofaltered, non-pathogenic HSV-1 virus (as well as other viruses) as avector for gene therapy. Other aspects of the present invention relateto assays for detecting candidate substances capable of acting asanti-apoptotic agents, as well as assays for detecting candidatesubstances able to induce programmed cell death in tumor cells.Additionally, the present invention also relates to methods for treatingcancer and other tumorgenic diseases. Finally, the present inventionalso relates to the use of candidate substances capable of inactivatingthe γ₁34.5 gene or ICP34.5 and thereby suppressing HSV-1 and other viralinfections.

The wild-type HSV-1 genome (150 kilobase pairs) has two components, Land S, each possessing unique sequences flanked by inverted repeats. Therepeat sequences of the L component, designated ab and b′a′, are each 9kilobase pairs, whereas the repeat sequences of the S component,designated a′c′ and ca, are each 6.5 kilobase pairs. Wadsworth et al.,J. Virol., 15: 1487-1497 (1975). The shared a sequence, 500-base pairslong in HSV1 strain F [HSV-1(F)], is present in one copy at the Scomponent terminus and in one to several copies, in the sameorientation, at the junction between L and S components. The L and Scomponents invert relative to each other such that the DNA extractedfrom virions or infected cells consists of four isomers differing solelyin the orientation of the L and S components relative to each other.Hayward et al., Proc. Natl. Acad. Sci. USA, 72: 4243-4247 (1975). The asequence appears to be a cis-acting site for inversions inasmuch asinsertion of the a sequence elsewhere in the genome or deletion of theentire internal inverted repeat sequences (b′a′c′) leads to additionalinversions or the loss of the ability of the L and S components toinvert, respectively. The a sequence was also shown to contain thecis-acting sites for the circularization of the genome after infection,for cleavage of the HSV genome from concatemers, and for encapsidationof the DNA.

HSV-1 genomes contain at least 73 genes whose expression is coordinatelyregulated and sequentially ordered in cascade fashion. The a genes areexpressed first, followed by β, γ₁ and γ₂ genes. The differentiationamong β, γ₁ and γ₂ genes is operationally based on the effect ofinhibitors of viral DNA synthesis. Whereas the expression of β genes isstimulated and that of γ₁ genes is only slightly reduced by inhibitorsof viral DNA synthesis, the expression of γ₂ genes stringently requiresviral DNA synthesis.

In the course of studies on the function of the a sequence, Chou andRoizman (Cell, 41: 803-811, 1985) noted that the chimeric structureconsisting of the a sequence fused to the 5′ transcribed, noncodingsequences of the thymidine kinase (TK) gene of HSV-1 was inducible intransferred cells and regulated as a γ₁ gene when inserted into theviral genome. This observation suggested that the terminus of the asequence nearest the b sequence of the inverted repeats contained apromoter and the transcription initiation site of a gene whosestructural sequences were located in the b sequences flanking the Lcomponent. Studies involving hybridization of labeled DNA probes toelectrophoretically separated RNAs extracted from infected cells, and S1nuclease analyses confirmed the existence of RNA transcripts initiatingin the a sequence. Nucleotide sequence analyses revealed the presence ofan open reading frame capable of encoding a protein 263 amino acidslong. Chou and Roizman, J. Virol., 64: 1014-1020 (1990).

Previo us studies have shown that each inverted repeat of the Scomponent contains in its entirety a gene designated α4, whereas each ofthose of the L component contains in its entirety a gene designated α0.See, e.g., Mackem and Roizman, J. Virol., 44: 934-947 (1982). Theputative gene identified on the basis of nucleotide sequence andanalyses of RNA is also present in two copies per genome. Because of theoverlap of the domain of this gene with the a sequence containing thecis-acting sites for inversion, cleavage of DNA from concatemers, andpackaging of the DNA, it was of interest to identify and characterizethe gene product. For this purpose, the observation that the nucleotidesequence predicted the presence in the protein of the amino acid tripletAla-Thr-Pro repeated 10 times was utilized, and antibody to a syntheticpeptide synthesized on the basis of this sequence reacted with a43,500-apparent-molecular-weight HSV-1 protein. Ackerman et al., J.Virol., 58: 843-850, 1986.

The extent of variability of the open reading frame that encodes ICP34.5was established by compar ing the nucleotide sequences of thr e e HSV-1strains passaged a limited number of times outside a human host. Chouand Roizman (J. Virol., 64: 1014-1020, 1990) reported that the gene thatspecifies ICP34.5 contains 263 codons conserved in all three limitedpassage strains but not in the reported sequence of the HSV-1(17)syn+strain. (FIG. 1 a-1 d) To ensure that the antibody to a predicted repeatsequence, Ala-Thr-Pro, reacted with ICP34.5 rather than with aheterologous protein with a similar repeat sequence, a short sequence of45 nucleotides that encodes an epitope characteristic of another HSV-1gene was inserted near the 5′ terminus of the ICP34.5-coding domain. Therecombinant virus expressed a protein with an appropriately slowerelectrophoretic mobility and which reacted with both the monoclonalantibody to the inserted epitope and rabbit antiserum to the Ala-Thr-Prorepeat element.

Studies of the identification of the genes associated withneurovirulence have repeatedly implicated DNA sequences located at ornear a terminus of the long component of HSV-1 DNA. Thus,Centifanto-Fitzgerald et al. (J. Exp. Med., 155: 475,1982) transferred,by means of a DNA fragment, a virulence marker from a virulent to anantivirulent strain of HSV-1. Deletion of genes located at one terminusof the long component of HSV-1 DNA contributed to the lack of virulenceexhibited by a prototype HSV vaccine strain. Meignier et al., J. Infect.Dis., 158: 602 (1988). In other studies, Javier et al. (J. Virol., 65:1978, 1987) and Thompson et al. (Virology, 172: 435, 1989) demonstratedthat an HSV-1×HSV-2 recombinant virus consisting largely of HSV-1 DNAbut with HSV-2 sequences located at one terminus of the long componentwas avirulent; virulence could be restored by rescue with the homologousHSV-1 fragment. Taha et al. (J. Gen. Virol., 70: 705, 1989) described aspontaneous deletion mutuant lacking 1.5 kbp at both ends of the longcomponent of a HSV-2 strain. Because of heterogeneity in the parentvirus population, the loss of virulence could not be unambiguouslyrelated to the specific deletion, although the recombinant obtained bymarker rescue was more virulent than the deletion mutant. In neitherstudy was a specific gene or gene product identified at the mutatedlocus, and no gene was specifically linked to virulence phenotype.

Role of the γ₁134.5 Gene

To test the possible role of the product of the γ₁34.5 gene, ICP34.5, aseries of four viruses (FIG. 2) were genetically engineered by theprocedures of Post and Roizman (Cell, 25: 227, 1981, incorporated hereinby reference).

1) Recombinant virus R4002 (FIG. 4 a, lane 3 and FIG. 4 b, lane 3)contained the insertion of a thymidine kinase (tk) gene driven by thepromoter of the α27 gene (α27-tk) in both copies of the ICP34.5 codingsequences. It was constructed by cotransfecting rabbit skin cells withintact DNA of HSV-1(F)Δ305, a virus from which a portion of the tk genewas specifically deleted, with the DNA of plasmid pRB3615, whichcontains the α27-tk gene inserted into the γ₁34.5 gene contained in theBamHI S fragment. Recombinants that were tk⁺ were then selected on human143 thymidine kinase minus (TK⁻) cells. The fragment containing theα27-tk gene contains downstream from the tk gene: the 5′ untranscribedpromoter, the transcribed noncoding sequence, and the initiatingmethionine codon of the glycoprotein H gene. The BstEII site into whichthe α27-tk fragment was inserted is immediately upstream of the codon 29of the γ₁34.5 open reading frame. As a consequence, the initiating codonof glycoprotein H was fused in frame and became the initiating codon ofthe truncated open reading frame of the γ₁34.5 gene (FIG. 2, line 3).The recombinant selected for further study, R4002, was shown to containthe α27-tk gene insert in both copies of γ₁34.5 gene (FIG. 3 a, lane 4and FIG. 3 b, lane 4) and specified only the predicted truncated productof the chimeric γ₁34.5 gene (FIG. 4 b). The amounts of the nativeICP34.5 protein detected in these and previous studies have beengenerally low. The chimeric genes formed by the fusion of the 5′transcribed noncoding region and the initiating codon of glycoprotein Hin frame with the truncated γ₁34.5 gene were expressed far moreefficiently than the native genes.

2) The recombinant virus R3617 (FIG. 2, line 5 from the top) lacking 1kb of DNA in each copy of the γ₁34.5 gene was generated bycotransfecting rabbit skin cells with intact R4002 DNA and the DNA ofplasmid pRB3616. In this plasmid, the sequences containing most of thecoding domain of γ₁34.5 has been deleted (FIG. 2, line 5 from top). Thetk⁻ progeny of the transfection was plated on 143TK⁻ cells overlaid withmedium containing bromodeoxy uridine (BrdU). This procedure selects tk⁻viruses, and since the tk gene is present in both copies of the γ₁34.5gene, the selected progeny of the transfection could be expected tocontain deletions in both copies. The selected tk⁻ virus designated asR3617 was analyzed for the presence of the deletion in both copies ofthe γ₁34.5 gene. For assays of neurovirulence, the deletion in thenative tk⁻ gene of R3617, which traces its origin from HSV-1(F)Δ305, hadto be repaired. This was done by cotransfection of rabbit skin cellswith intact R3617 DNA and BamHI Q fragment containing the tk gene. Thevirus selected for tk⁺ phenotype in 143TK⁻ cells was designated R3616.This virus contains a wild-type BamHI Q fragment (FIG. 3 b lane 6) anddoes not make ICP34.5 (FIG. 4 b).

3) To ascertain that the phenotype of R3616 indeed reflects the deletionin the γ₁34.5 gene, the deleted sequences were restored bycotransfecting rabbit skin cells with intact R3617 DNA, the HSV-1(F)BamHI Q DNA fragment containing the intact tk gene, and the BamHI SP DNAfragment containing the intact γ₁34.5 gene in the molar ratios of1:1:10, respectively. Viruses that were tk⁺ were then selected in 143TK⁻cells overlaid with medium containing hypoxanthine, aminopterin, andthymidine. The tk⁺ candidates were then screened for the presence ofwild-type tk and γ₁34.5 genes. As expected, the selected virusdesignated HSV-1(F)R (FIG. 2, line 6) contained a wild-type terminallong component fragment (compare FIG. 3 a, lanes 2, 7, and 8), andexpressed ICP34.5 (FIG. 4 a, lane 6).

4) To eliminate the possibility that the phenotype of R3616 reflectsdeletion in cryptic open reading frames, a virus was constructed (R4010,FIG. 2, line 7 from the top) that contains translational stop codons inall three reading frames in the beginning of the ICP34.5 codingsequence. The 20-base oligonucleotide containing the translational stopcodons and its complement sequence (FIG. 2) were made in an AppliedBiosystems 380D DNA synthesizer, mixed at equal molar ratio, heated to80° C., and allowed to cool slowly to room temperature. The annealed DNAwas inserted into the HSV-1(F) BamHI S fragment at the BstEII site. Theresulting plasmid pRB4009 contained a stop codon inserted in thebeginning of the ICP34.5 coding sequence. The 20 nucleotide oligomer DNAinsertion also contained a Spe I restriction site, which allowed rapidverification of the presence of the insert. To generate the recombinantvirus R4010, rabbit skin cells were cotransfected with the intact DNA ofR4002 and the pRB4009 plasmid DNA. Recombinants that were tk⁻ wereselected in 143TK⁻ cells in medium containing BrdU. The tk⁺ version ofthis virus, designated R4009, was generated by cotransfection of intacttk⁻ R4010 DNA with HSV-1(F) BamHI Q DNA fragment, and selection of tk⁺progeny. The virus selected for neurovirulence studies, R4009, containedthe Spe I restriction endonuclease cleavage site in both BamHI S and SPfragments (compare FIG. 3 a, lanes 9 and 10) and did not express ICP34.5(FIG. 4 b, lane 7).

5) R4004 (FIG. 2, last line) was a recombinant virus produced byinsertion of a sequence encoding 16 amino acids. This sequence has beenshown to be the epitope of the monoclonal antibody H943 reactive with aviral protein designated as ICP4. Hubenthal-Voss et al., J. Virol., 62:454 (1988). The virus was generated by cotransfecting intact R4002 DNAand the DNA of plasmid pRB3976 containing the insert, and the selectedtk-progeny was analyzed for the presence of the insert. Forneurovirulence studies, its tk gene was restored (recombinant virusR4003) as described above. The DNA sequence was inserted in frame at theNco I site at the initiating methionine codon of the γ₁34.5 gene. Theinsert regenerated the initiating methionine codon and generated amethionine codon between the epitope and the remainder of ICP34.5.Because of the additional amino acids, the protein migrated more slowlyin denaturing polyacrylamide gels (FIG. 4 b, lane 4).

Plaque morphology and size of all of the recombinants were similar tothose of the wild-type parent, HSV-1(F) when plated on Vero, 143TK⁻, andrabbit skin cells lines. Whereas HSV-1(F)R and R4003 replicated as wellas the wild-type virus in replicate cultures of Vero cells, the yieldsof R3616 and R4009 were reduced to one-third to one-fourth the amount ofthe wild type. Although ICP34.5 was not essential for growth of HSV-1 incells in culture, the results of the studies shown in Table 1 indicatethat the deletion or termination of translation of the γ₁34.5 has aprofound effect on the virulence of the virus. Thus, all of the miceinoculated with the highest concentration [1.2×10⁶ plaque-forming units(PFU)] of R3616 survived. In the case of R4009, only three of ten micedied as a result of inoculation with the highest concentration of virus(˜10⁷ PFU). In comparison with other deletion mutants, R3616 and R4009rank among the least pathogenic viruses reported to date. The virus inwhich the γ₁34.5 gene was restored exhibited the virulence of the parentvirus.

TABLE 1 Virus in the inoculum Genotype PFU/LD₅₀ HSV-1(F) Wild-typepartent virus 420 R3616 1000-bp deletion in the _(γ1)34.5 >1,200,000HSV-1(F)R Restoration of _(γ1)34.5 and tk 130 R4009 Stop codon in_(γ1)34.5 >10,000,000 R4003 Monoclonal antibody epitope 4,200 insertedat the NH₂ terminal Comparative ability of wild-type and recombinantviruses to cause death after intracerebral inoculation of mice. Theneurovirulence studies were done on female BALB/C mice obtained at 21days of age (weight ± 1.8 g) from Charles River Breeding Laboratories inRaleigh, North Carolina. The viruses were diluted in minimum essentialmedium containing Earle's salts and 10% fetal bovine serum, penicillin,and gentamicin. The mice were inoculated intracerebrally #in the rightcerebral hemisphere with a 26-gauge needle. The volume delivered was0.03 ml, and each dilution of virus was tested in groups of ten mice.The animals were checked daily for mortality for 21 days. The LD₅₀ wascalculated with the aid of the “Dose effect Analysis” computer programfrom Elsevier Biosoft, Cambridge, United Kingdom.

The wild-type virus and all of the recombinants have identical surfaceglycoproteins necessary for attachment and penetration into brain cells.Injection of 106 PFU into the brain should result in infection and deathof a significant number of the brain cells. Death after intracerebralinoculation results from viral replication, spread from cell to cell,and cell destruction before the immune system has a chance to act.Titrations of brain tissue suspended in minimal essential mediumcontaining Eagle's salts and 10% fetal bovine serum showed that thebrains of animals inoculated with the viruses that failed to makeICP34.5 contained very little virus. Thus, for the R3616 and R4009viruses, the recovery was 120 and 100 PFU per gram of brain tissue,respectively. Given the amount of virus in the inoculum (highestconcentration tested), it is not clear whether the small amounts ofrecovered virus represent a surviving fraction of the inoculum or newlyreplicated virus. In contrast, the amounts of virus recovered from miceinoculated with HSV-1(F)R and R4003 were 6×10⁶, respectively. Theseresults indicate that the failure of the two recombinant viruses tocause death must be related to poor spread of virus in neuronal tissueas a consequence of the inability of mutant viruses to replicate in theCNS, reflecting a reduction in their host range.

In the course of studies designed to determine the function of theγ₁34.5 gene product, it was discovered that infection of cells ofneuronal origin with mutants incapable of expressing the γ₁34.5 generesulted in shutoff of cellular protein synthesis, whereas infection ofcells of non neuronal origin with wild type or mutant viruses resultedin sustained protein synthesis and production of infectious progeny.

EXAMPLE 1 IMPACT OF γ₁34.5 EXPRESSION ON PROGRAMMED CELL DEATH Materialsand Methods

Cells

Vero cells originally obtained from ATCC were propagated in DME mediacontaining 5% calf serum. The human SK-N-SH neuroblastoma (NB) cell linewas obtained from ACTT (HTB11)′ and propagated in Dulbecco's modifiedEagle's medium containing 10% fetal bovine serum.

Viruses

The isolation of herpes simplex virus 1 strain F, [HSV-1(F)] has beendescribed by Ejercito et al. (J. Gen. Virol., 2: 357-364 (1968)(incorporated herein by reference). The construction of recombinantviruses R3616, R4009, and HSV-1(F) was reported by Chou et al. (Science,250: 1262-1266, Nov. 30, 1990) (incorporated herein by reference). Asillustrated in FIG. 5, R3616 contains a 1 Kbp deletion in both copies ofthe γ₁34.5 gene. In the recombinant R4009 a stop codon was inserted inboth copies of the γ₁34.5 gene. The γ₁34.5 genes in the recombinantR3616 were restored to yield the recombinant HSV-1(F)R.

Virus Infection

Cells were generally exposed to the viruses for 2 h at 37° C. atmultiplicity of infection of 5 and then removed and replaced with the199v media containing 1% calf serum. The infection continued at 37° C.for a length of time as indicated for each experiment. Cells were theneither labeled for de novo protein synthesis or analysis of viral DNAand RNA.

³⁵S-methionine labeling

At the indicated time post infection, 50 uCi of ³⁵S-methionine (specificactivity >1,000 Cimmole, Amersham Co., Downers Grove, Ill.) was added to1 ml of 199v media lacking methionine to cells in 6 well dishes.Labeling was continued for 1.5 hr, at which time cells were harvested.Preparation of cell extracts; separation of proteins by electrophoresisin denaturing polyacrylamide gels crosslinked with N,N′Diallytartardiamide (Bio-Rad Laboratories, Richmond, Calif.); transferof polypeptides to nitrocellulose sheets; autoradiography and immunoblotwith antibodies have been described by Ackermann et al., J. Virol., 52:108-118 (1984) (incorporated herein by reference).

Results

HSV-1 recombinant viruses lacking the γ₁34.5 gene induce the shut offprotein synthesis in neuroblastoma cells. In the course of screeninghuman cell lines derived from CNS tissues it was apparent that the SK-NSH neuroblastoma cell lines produced 100 fold less mutant viruses thanthe fully permissive Vero cells. In was also noted that the SK-N-SHneuroblastoma cells infected with R3616 or with R4009 exhibited reducedprotein synthesis in cells harvested at 7 hours (FIG. 6 a) and ceased toincorporate ³⁵S-methionine by 13 hours (FIG. 6 b) post infection. Thephenomenon was observed in SK-N-SH neuroblastoma cells only, and couldbe attributed specifically to the mutations in the γ₁34.5 gene inasmuchas restoration of the deleted sequences yielded a virus [HSV-1(F)R]which expressed viral proteins at 13 hour post infection (FIG. 6 b) andexhibited the parental, wild type phenotype.

The shut off of protein synthesis occurred after the expression of agenes. Viral genes form three major groups whose expression iscoordinately regulated and sequentially ordered in a cascade fashion.See Roizman and Sears, Fields' Virology, 2 ed., Fields et al., Eds,1795-1841 ( 1990 ). The a genes do not require de novo protein synthesisfor their expression, the β genes which are required for the synthesisof viral DNA require prior synthesis of functional α and β proteins andthe onset of viral DNA synthesis. To determine the point at whichexpression of viral gene functions was terminated in SK-N-SHneuroblastoma cells infected with mutant viruses, infected cell lysateselectrophoretically separated in denaturing polyacrylamide gels weretransferred to a nitrocellulose sheet and probed with antibody to an a(α27), a β (viral thymidine kinase) and two abundant γ proteins. Rollerand Roizman (J. Virol., 65: 5873-5879 (1991) have shown that the latterwere the products of U_(L)26.5 and of U_(S)11 genes whose expression atoptimal levels requires viral DNA synthesis. As shown in FIGS. 7 a-7 c,the SK-N-SH neuroblastoma cells infected with the mutant viruses madenormal amounts of α27 protein (FIG. 7 a), reduced amounts of thethymidine kinase (β) protein (FIG. 7 b), but no detectable γ proteins(FIGS. 7 a and 7 b). In contrast, both the wild type and mutant virusescould not be differentiated with respect to their capacity to replicateor to direct the synthesis of their proteins in Vero cells (FIGS. 7 a-7c).

The signal for shut off of protein synthesis is linked to viral DNAsynthesis. These experiments were designed to determine whether theshutoff of protein synthesis was linked to a gene whose expression wasdependent on viral DNA synthesis. The results of a key experiment areshown in FIG. 8. Replicate SK-N-SH and Vero cell cultures were infectedwith HSV-1(F) and recombinant viruses and maintained in the presence orabsence of inhibitory concentrations of phosphonoacetate, a drug whichblocks viral DNA synthesis. The cells were pulse labeled withS³⁵-methionine at 13 h post infection. The salient feature of theresults were that protein synthesis in SK-N-SH cells infected witheither R3616 or R4009 was sustained for at least 13 h in the presence ofPhosphonoacetate but not in its absence. These results indicted that thesignal for cessation of protein synthesis in SK-N-SH neuroblastoma cellsinfected with mutant viruses was associated with viral DNA synthesis orwith a γ gene dependent on viral DNA synthesis for its expression.

Human neuroblastoma cells infected with the γ₁34.5⁻ mutants synthesizedviral DNA and accumulated late mRNA even though the shut off of proteinsynthesis precluded accumulation of late proteins. The evidencepresented above indicated that in SK-N-SH neuroblastoma cells an eventassociated with viral DNA synthesis triggered the shut off of proteinsynthesis and that the late (γ) viral proteins did not accumulate. Weexpected, therefore, little or no accumulation of viral DNA and in theabsence of viral DNA synthesis, little or no accumulation of late (γ))viral transcripts whose synthesis is dependent on viral DNA synthesis.To our surprise, the amounts of viral DNA recovered from SK-N-SHneuroblastoma cells 17 hours post infection with mutant viruses werecomparable to those obtained from wild type parent or repaired[(HSV-1)F)R] viruses (FIG. 9 a). Furthermore, while the SK-N-SHneuroblastoma cells did not synthesize demonstrable amounts of U_(S)11protein, the amounts of U_(S)11 gene transcripts which accumulated incells infected with mutant and wild type viruses were of similarmagnitude (FIG. 9 b).

The significance of these results stems from three observations. First,in infected cells, protein synthesis reflects a regulatory cascade; aprotein synthesis is replaced by β and later by γ protein synthesis. Inall cell lines other than the SK-N-SH neuroblastoma cells infected withthe γ₁34.5 mutants and tested to date, a block in the synthesis of onegroup of proteins does not lead to a cessation of total proteinsynthesis. For example, in cells treated with inhibitors of DNAsynthesis like PAA, a subclass of γ proteins dependent for theirsynthesis on viral DNA synthesis is not made. However, in these cells, βprotein synthesis continues beyond the time of their synthesis inuntreated infected cells. The striking observations made in the studieson SK-N-SH cells infected with the γ₁34.5 null mutants are that (i) allprotein synthesis ceased completely, (ii) viral DNA was made and (iii)late, γ mRNA exemplified by U_(S)11 mRNA was made even though proteinsynthesis ceased. These manifestations for viral replication have notbeen reported previously and are not characteristic of cells infectedwith wild type virus or any mutant virus infection of cells (e.g. Vero,HEp-2, baby hamster kidney, 143tk⁻ and rabbit skin cell lines and humanembryonic lung cells strain) other than those described in this report.

Second, the function of the γ₁34.5 gene is to overcome this block inprotein synthesis in SK-N-SH cells since repair of the mutation restoresthe wild type phenotype.

Lastly, while the association of cessation of protein synthesis with theonset of viral DNA replication does not exclude the possibility that aproduct made after infection is responsible of the shut off, the datadoes support the hypothesis that the cessation of protein synthesis isspecifically caused by a known viral gene product interacting with theprotein synthesizing machinery of the cell. For example, it has beenwell established that the product of the HSV-1 gene designated a vhs canshut off cellular protein synthesis after infection. vhs is a structuralprotein of the virus and is introduced into cells during infection. Itdestabilizes mRNA early in infection and its effects are not dependenton viral gene products made after infection. In the experiments setforth above, protein synthesis of wild type and mutant viruses could notbe differentiated at 13 hours post infection in cells treated withPhosphonoacetate and hence the phenotype of mutant viruses cannot beattributed to the vhs gene product. This conclusion is reinforced by theobservation that viral protein synthesis in SK-N-SH cells was notaffected by increasing the multiplicity of infection with wild typevirus to values as high as 100 pfu/cell (data not shown). A more likelysource for the genetic information for the cessation of proteinsynthesis is the cell itself.

It has been reported that deprivation of growth factors from cells ofneuronal origins results in programmed cell death, which manifestsitself initially by the cessation of protein synthesis and subsequentlyby fragmentation of DNA. Apoptosis in lymphocytes is manifested bydegradation of DNA. In the case of other herpes viruses, it has beenshown that in B lymphocytes infected with the Epstein-Barr virus, theproduct of the viral IMP-1 gene induces the host gene Bcl-2 whichprecludes programmed lymphocyte death (Henderson et al, Cell 65:1107-1991. Thus, it is apparent that the onset of viral DNA synthesis inneuronal cells triggers programmed cell death by cessation of proteinsynthesis and that HSV-1 γ34.5 gene precludes this response.

The evolution of a HSV-1 gene which would preclude a response to aneuronal stress is not surprising. Infection of neurons, especiallysensory neurons, is an essential feature of viral reproductive lifestylewhich enables the HSV-1 to remain latent and to survive in humanpopulations. If, as we propose, the function of γ₁34.5 gene is topreclude cell death, the target of the gene would be neurons rather thanlymphocytes since HSV normally infects nerve cells.

The γ₁34.5 gene has several unusual features. The gene lacks aconventional TATAA box or response elements frequently associated withTATAA-less transcriptional units. The sequence which enables theexpression of the gene is 12 bp long but repeated as many as 3 times inthe wild type strain used in this laboratory. Various assays reportedelsewhere indicate that the amounts of gene products produced in cellsof non neuronal derivation are smaller than those expressed by mostviral genes and that the amounts of the protein made in the absence ofviral DNA synthesis were smaller than those made in its presence. Thegene is predicted to encode a protein of 263 amino acids. It containsthe triplet Ala-Thr-Pro repeated 10 time and accumulates in thecytoplasm. A recent note indicates that 63 amino acid residues near thecarboxyl terminus of the γ₁34.5 protein shares 83% identity with a mouseprotein MyD116 found in a myeloid leukemic cell line induced todifferentiate by interleukin 6 (Lord et al., Nucleic Acid Res. 18: 2823,1990). The function of MyD116 is unknown. The results presented abovedemonstrate that the product of the γ₁34.5 gene, the protein ICP34.5,quite clearly enables sustained protein synthesis in SK-N-SHneuroblastoma cells, and it is clear that the gene's expression issufficient to preclude apoptosis.

The promoter-regulatory elements essential for the expresion of γ₁34.5are contained within three elements of the a sequence, i.e. the directrepeats DR2 and DR4 and the unique U_(b) sequences. Gel retardationassays failed to show binding of the product of the α4 gene encoding themajor regulatory protein of the virus to any of the elements regulatingexpression of the γ₁34.5 gene. In transient expression assays, theproduct of the α4 or of α0 genes failed to transactivate a chimericreporter gene consisting of the coding sequences of the thymidine kinasegene fused to the 5′ non-coding sequences of the γ₁34.5 gene. Thereporter gene was induced, but to a relatively low level byco-transfection with plasmids containing both α4 and α0 genes. Theplasmid encoding the α27 gene had no effect on the expression of thechimeric reporter gene transfected alone although it reduced theinduction of the chimeric gene by plasmids containing the α0 and α4genes.

EXAMPLE 2 TREATMENT OF PROGRAMMED CELL DEATH (APOPTOSIS) WITH GENETHERAPY

In this example, γ₁34.5 gene therapy directed toward the prevention ortreatment of apoptosis is described. For the purposes of this examplemutated HSV-1 virus is proposed as a vector for introduction of the geneinto neuronal cells undergoing or about to undergo programmed celldeath. It is also envisioned that this embodiment of the presentinvention could be practiced using alternative viral or phage vectors,including retroviral vectors and vaccinia viruses whose genome has beenmanipulated in alternative ways so as to render the virus nonpathogenic.The methods for creating such a viral mutation are set forth in detailin U.S. Pat. No. 4,769,331, incorporated herein by reference.Furthermore, it is also envisioned that this embodiment of the presentinvention could be practiced using any gene whose expresion isbeneficial in gene therapy, and use of the non-HSV viruses would allowgene therapy in non-neural systems.

Herpes simplex virus has a natural tropism for human CNS tissue. Underwild type conditions, the virus is capable of replicating andmultiplying in the nervous system and is neurovirulent. The virus canalso establish latent infection in the neurons and can be occasionallyreactivated. To establish a vector system for delivery of genes intoneurons, the proposed construct of an HSV vector must satisfy thefollowing criteria: 1. Such a vector should have a natural tropism forCNS and brain tissue. 2. Such a vector should be non-pathogenic; thatis, totally avirulent and not reactivatable to cause an infection. 3.Such a vector should consist of constitutive expression of γ₁34.5 toprevent cell death in cells undergoing neurodegeneration. 4. Such avector so proposed in 3 is suitable for additional foreign geneinsertion for gene therapy.

Material and Methods

A HSV vector with a mutational lesion in the α4 gene is constructed. Theproposed virus will no longer be able to replicate, multiply andreactivate from latent infection in the CNS. The virus can, in theabsence of α4 gene, establish a latent infection in the neuron. Thisvirus can be obtained by co-transfection of viral DNA with plasmidcontaining a α4 expressing cell line. α4 expressing cell lines and thevirus have been reported previously. DeLuca et al., J. Virol., 56:558-570 (1985).

Additionally, such an HSV vector with γ₁34.5 gene under a constitutiveexpression promoter is also envisioned. This constitutive expressionpromoter can be the HSV LAT promoter, the LTR promoter of retrovirus orany other foreign promoter specific for neural gene expression. Such aviral vector properly introduced is suitable for prevention of celldeath in neuronal cells undergoing apoptosis.

Moreover, such an HSV vector with foreign genes inserted at a neutrallocation on the viral genome is suitable for delivery of foreign genesinto target neurons and for CNS gene therapy. The procedures to generatethe above recombinant viruses are those published by Post and Roizman(Cell, 25: 227, 1991) incorporated herein by reference. See also U.S.Pat. No. 4,769,331, incorporated herein by reference.

In instances where use of the mutated HSV-1 virus is appropriate thevirus can be combined with a pharmaceutically acceptable carrier such asbuffered saline and injected at the site of peripheral nerve endingswhose axons originate from neural cell bodies undergoing or about toundergo apoptosis. As will be recognized by those skilled in the medicalarts the amount of virus administered will vary depending upon severalfactors, including the vector's ability to target the cells requiringtreatement, the extent to which the gene is expressed in the targettissue, and the activity of the expressed protein, among others. Aninnoculum containing approximately 10⁴-10⁵ viruses in phosphate bufferedsaline or skim milk has produced successful results in mice. Virus soinjected is taken up into the peripheral nerve endings and is thentransported via retrograde axonal transport to the neuronal cell bodies.In instances where such peripheral injection is not useful orappropriate, localized intraspinal or intraventricular injection, ordirect microinjection of the virus could be utilized.

An appropriately altered non-HSV virus, one with a genome manipulated insuch a way as to render the virus non-pathogenic, could be used in asimilar manner. Direct microinjection or peripheral injection fordelivery to the cell body via retrograde axonal transport are optionsfor viral delivery. Finally, it should also be noted that a biologicalfunctional equivalent gene could be utilized for gene therapy in anyvector described in this example.

EXAMPLE 3 USE OF MULTI-POTENT NEURAL CELL LINES TO DELIVER THE γ₁34.5GENE TO THE CNS

In addition to the viral vector delivery system to CNS and brain tissue,another vector system has been developed recently using cell linespassaged in vitro and engrafting these cells back to the animal. Theseprocedures involve taking cells of fetal or postnatal CNS origin,immortalizing and transforming them in vitro and transplanting the cellsback into the mouse brain. These cells, after engraftment, follow themigration pattern and environmental cue of normal brain cell developmentand differentiate in a nontumorigenic, cytoarchitecturally appropriatemanner. This work has been examplified in several articles notablySnyder et al., Cell, 68: 33-51, 1992 and Ranfranz et al., Cell, 66:713-729, 1991. Utilizing appropriately modified techniques, it ispossible to introduce the γ₁34.5 gene alone or in combination with othergenes of interest into the cells and engraft. Such a procedure allowsthe delivery of the genes to its natural site. Proper expression of theγ₁34.5 gene in these neurons should result in prevention of cell deathin neurodegeneration and preserving cells carrying foreign genessuitable for gene therapy.

Materials and Methods

Propagation of Cerebellar Cell Lines

Cerebellar cell lines are generated as described by Ryder et al. (J.Neurobiol. 21: 356-375, 1990). Lines are grown in Dulbecco's modifiedEagle's medium supplemented with 10% fetal calf serum (Gibco), 5% horseserum (Gibco), and 2 mM glutamine on poly-L-lysine (PLL) (Sigma) (10μgml)-coated tissue culture dishes (Corning). The lines are maintainedin a standard humidified, 37° C., 5% CO₂-air incubator and are eitherfed weekly with one-half conditioned medium from confluent cultures andone-half fresh medium or split (1:10 or 1:20) weekly or semiweekly intofresh medium.

Transduction of Cerebellar Progenitor Lines with γ₁34.5 Gene

A recent 1:10 split of the cell line of interest is plated onto 60 mmtissue culture plates. Between 24 and 48 hr after plating, the cells areincubated with the replication-incompetent retroviral vector BAGcontaining the −myc gene (10⁶-10⁷ colonyforming units [cfu]/ml) plus 8μg/ml polybrene for 1-4 hr for introduction of the γ₁34.5 gene alone orin combination with other suitable genes for gene therapy, along withthe neomycin G418 marker. Cells are then cultured in fresh feedingmedium for approximately 3 days until they appear to have undergone atleast two doublings. The cultures are then trypsinized and seeded at lowdensity (50-5000 cells on a 100 mm tissue culture dish). Afterapproximately 2 weeks well-separated colonies are isolated by briefexposure to trypsin within plastic cloning cylinders. Colonies areplated in 24-well PLL-coated CoStar plates. At confluence, thesecultures are passaged to 60 mm tissue culture dishes and expended. Arepresentative dish from each subclone is stained directly in theculture dish using X-gel histochemistry (see Price et al., 1967; Cepko,1989a, 1989b). The percentage of blue cells is determined under themicroscope. Subclones with the highest percentage of blue cells(ideally >90%; at least >50%) are maintained, characterized, and usedfor transplantation.

Tests for Virus Transmission

The presence of helper virus is assayed by measurement of reversetranscriptase activity in supernatants of cells lines as described byGoff et al. (1981) and by testing the ability of supernatants to infectNIH 3T3 cells and generate G418-resistant colonies of X-gal⁺ colonies(detailed in Cepko, 1989a, 1989b). All cerebellar cell lines used fortransplantation are helper virus-free as judged by these methods.

Coculture of Neural Cell Lines with Primary Cerebellar Tissue

Primary dissociated cultures of neonatal mouse cerebellum are preparedas in Ryder et al. (1990) and seeded at a density of 2×10⁶ to 4×10⁶cells per PLL-coated eight-chamber Lab Tek glass or plastic slide(Miles). After the cells settled (usually 24 hr), 10% of a nearlyconfluent 10 cm dish of the neural cell line of interest is seeded,following trypsinization, onto the slide. The coculture is re-fed everyother day and grown in a 50% CO₂-air, humif ied incubator until 8 or 14days of coculture.

Preparation of Cells Lines for Transplantation

Cells from a nearly confluent but still actively growing dish of donorcells are washed twice with phosphate-buffered saline (PBS),trypsinized, gently triturated with a wide-bore pipette inserum-containing medium (to inactivate the trypsin), gently pelleted(1100 rp for 1 min in a clinical centrifuge), and resuspended in 5 ml ofPBS. Washing by pelleting and resuspension of fresh PBS is repeatedtwice, with the cells finally resuspended in a reduced volume of PBS toyield a high cellular concentration (at least 1×10⁶ cells per μl).Trypan blue (0.05% w/v) is added to localize the inoculum. Thesuspension is kept well triturated, albeit gently, and maintained on iceprior to transplantation to minimize clumping.

Injections into Postnatal Cerebellum

Newborn CD-1 or CF-1 mice are cryoanesthetized, and the cerebellum islocalized by transillumination of the head. Cells are administeredeither via a Hamilton 10 μl syringe with a beveled 33-gauge needle or adrawn glass micropipette with a 0.75 mm inner diameter and 1.0 mm outerdiameter generated from borosilicate capillary tubing (FHC, Brunswick,Me.) by a Flaming Brown Micropipette Puller (Model p-87, SutterInstruments) using the following parameters: heat 750, pull 0, velocity60, time 0. Best results are achieved with the glass micropipette. Thetip is inserted through the skin and skull into each hemisphere andvermis of the cerebellum where the cellular suspension was injected(usually 1-2 μl per injection). Typically, the following situationshould exist: 1×10⁷ cells per ml of suspension; ×10⁶ to 2×10⁶ cells perinjection; one injection in each cerebellar hemisphere and in thevermis. Importantly, the cellular suspension, maintained on icethroughout, is gently triturated prior to each injection in order todiminish clumping and to keep cells suspended.

The injection of BAG virus was performed as described for the cellsuspension. The BAG virus stock (8×10⁷ G418-resistant cfu/ml) contained,in addition to trypan blue, polybrene at 8 μg/ml.

EXAMPLE 4 TREATMENT OF PROGRAMMED CELL DEATH (APOPTOSIS) WITH ICP34.5

As an alternative to the gene therapy methods described for exemplarypurposes in Examples 2 and 3, neuronal cells undergoing or about toundergo programmed cell death can also be treated with the proteinexpressed by the γ₁34.5 gene, i.e. ICP34.5. Alternatively, a biologicalfunctional equivalent protein could be used in such treatment.

For example, ICP34.5 is isolated from cells expressing the protein andpurified using conventional chromatography purification andimmunoaffinity purification methods described by Ackerman et al. (J.Virol. 58: 843-850, 1986, incorporated herein by reference). Thepurified protein is next combined with a pharmaceutically appropriatecarrier, such as buffered saline or purified distilled water. Foradministration, the pharmaceutical composition can be injected in one ofseveral ways, as appropriate: (i) intraspinal injection; (ii)intraventricular injection; (iii) direct injection into the areacontaining the neurons undergoing or about to undergo programmed celldeath or any other appropriate method of administration understood bythose skilled in the art. Such treatment would be particularlyappropriate in the surgical repair of severed peripheral nerves, and theuse of proteins as therapeutic agents is well within the current levelof skill in the medical arts in light of the present specification.

EXAMPLE 5 ASSAYS FOR CANDIDATE SUBSTANCES FOR PREVENTION OF PROGRAMMEDCELL DEATH (APOPTOSIS)

The γ₁34.5 gene of herpes simplex virus enables the virus to replicate,multiply and spread in the central nervous system and the brain so thatthe virus is neurovirulent to the host. Recombinant virus lacking thegene lost this ability to penetrate the CNS of the host and becometotally avirulent. In examining the nature of this avirulent phenotypein culture, the mutant virus lacking the gene exhibited a totaltranslation shutoff phenotype characteristic of programmed cell death.This mechanism of programmed cell death afforded by the host cellgreatly reduced the ability of the virus to multiply and spread. Thefunction of γ₁34.5 in the virus therefore is to inactivate theprogrammed death of the cell (anti-apoptosis) thereby restoringtranslation and enabling the virus to replicate to full potential in thehost.

This anti-apoptotic effect of γ₁34.5 was further examined and itsability to protect neural cells from other environmental stresses whichlead to apoptosis was discovered. These environmental stresses includeUV, nerve grown factor deprivation and neuronal cell differentiation.This Example describes the use of the γ₁34.5 gene and its protectivefunction to screen for pharmaceutical agents and drugs that mimic the invivo function of γ₁34.5 to prevent neurodegeneration. Such a screeningprocedure constitutes construction of cell lines expressing γ₁34.5 and anull cell line without the gene and measurement of the viability of thecells after stress treatment by induction of a reporter gene. This canbe a host gene promoter tagged by a fluorescence indicator or any othereasily assayable marker to signal viability.

Materials and Methods

A test neuroblastoma cell line is established constitutively expressingγ₁34.5 and containing a fluorescence tagged (e.g., the a sequencepromoter fused to lacZ) cellular gene, or any tag that provides theeasily assayable marker to signal viability. A neuroblastoma null cellline consisting of a-lacZ indicator gene and the same host indicatorgene is also established, along with a Vero cell line consisting ofa-lacZ indicator gene and the same host indicator gene. Environmentalstresses are then applied that normally would (1) trigger the a sequencepromoter activation; (2) trigger the protection afforded by γ₁34.5 assignaled by viability after stress treatment; and (3) trigger cellprogrammed death in the absence of γ₁34.5. Candidate substances ofpharmaceutically appropriate drugs and agents can be tested in such anassay. The proposed scheme of the assay for scoring of positivecandidates is shown in outline form in Table 2.

TABLE 2 EXPERIMENTAL FLOW CHART: ASSAY FOR CANDIDATE SUBSTANCE ABLE TOPREVENT PROGRAMMED CELL DEATH CELL LINES ACTION EXPECTATION A.neuroblastoma cells stress followed viability as constitutively expressby induction of measured by _(γ1)34.5 and a second second promoterinduction of inducible promoter- a reporter gene indicator gene hoursafter stress B. neuroblastoma cells stress followed 1. apoptosisexpressing a-lacZ by induction of related stress: and a second induciblesecond promoter a-lacZ induced; promoter second promoter not induced 2.toxicity: no induction of a-lacZ and the second induc- ible promoter C.vero cells stress followed 1. a-lacZ not expressing a-lacZ by inductioninduced and a second inducible promoter gene 2. toxicity factor exclu-ded, determined from expression of inducible second promoter

EXAMPLE 6 ASSAY FOR CANDIDATE SUBSTANCES FOR ACTIVATION OF PROGRAMMEDCELL DEATH (APOPTOSIS) FOR TREATMENT OF CANCER OR TUMOROGENIC DISEASESAND FOR SUPPRESSION OF HSV INFECTION

In order to induce cell death in tumor cells, it is desirable to blockthe expression of the anti-apoptosis gene or the activity of the proteinexpressed by the gene. As such, it is desirable to develop proceduresthat will allow screening for candidate substances which trigger celldeath in tumor cells. In addition, since expression of the γ₁34.5 geneof HSV-1 has been shown to prevent apoptosis in neuronal cells so thatthe virus can replicate, multiply and spread in the CNS (that is, sothat the virus can become neurovirulent), a substance capable ofblocking γ₁34.5 expression or inhibiting the action of ICP34.5can beexpected to supress HSV neurovirulence (or the virulence of otherviruses relying on a similar mechanism) by allowing apoptosis to occurin infected neurons.

It has been found that the protection afforded by γ₁34.5 can be extendedto protect other cells from environmental stresses, and indeed the genehas a generalized anti-apoptotic effect. The promoter for the geneγ₁34.5 lies in the a sequence of HSV and, at time of stress, thepromoter is activated. It can be assumed that the a sequence promotercontains apoptosis responsive elements and cellular factors(transcription factors in particular) that mediate the expression ofanti-apoptosis gene are apoptotic in nature. These cellular factors aretherefore the targets of the present assay to screen for drugs or agentsthat would inactivate their ability to induce the anti-apoptosis gene.The assay involves the use of the a sequence promoter and itsinducibility by conditions which induce apoptosis as an indicator assaywhich screens for therapeutic agents and drugs capable of blocking theexpression of the anti-apoptosis gene and therefore allow the cell todie of programmed cell death.

A test plasmid construct bearing the a sequence and coding sequence upto the 28th amino acid of γ₁34.5 is fused to the lacZ reporter gene orany other readily assayable reporter gene. The construct is introducedinto a neuroblastoma or PC12 cell line by G418 selection and a clonaland continuous cell line for screening purposes is established. Acontrol plasmid construct bearing an HSV late promoter, a promoter whichwould normally not be expressed in cell lines and which further wouldnot be induced to express by apoptosis-inducing stress is fused to thesame indicator gene. This construct is also introduced into a continuousclonal cell line and serves as a control for the test cell line.Environmental stresses that trigger the a sequence promoter activationand that cause programmed cell death are then defined. These conditionsinclude UV injury, virus infection, nerve growth factor deprivation, andthe influence of antibodies on cell surface receptors, among others.Candidate substances or pharmaceutically appropriate drugs and agentsare then tested in assays for their ability to block the a sequencepromoter activation at time of stress.

The assay of the present invention allows the screening andidentification of pharmaceutically appropriate drugs and agents targetedat various cellular factors that induce the expression of anti-apoptosisgene. By inactivating essential cellular factors, these agents should beable to allow cell programmed death to occur. Such positive candidateswould then be appropriately administered (via intravenous, intrathecal,or direct injection, or via oral administration) in order to induceprogrammed cell death in tumor cells, in neurons infected with theherpes virus, or in cells infected with a virus the virulence of whichis dependent upon an anti-apoptotic effect. The use of proteins andother chemotherapeutic substances in antitumor therapy is well known inthe art, and therefore, it is considered that the use and dosages ofcandidate substances for treatment of tumorogenic diseases (e.g.,cancer) or herpes infection is well within the skill of the presentstate of the medical arts in light of the present specification. SeeU.S. Pat. Nos. 4,457,916; 4,529,594; 4,447,355; and 4,477,245, allincorporated herein by reference. These positive candidates can also beused to identify intermediates in the pathways leading to cellprogrammed death.

Materials and Methods

W5 cell lines are established in 96 well culture dishes coated withcollagen. Control cell lines containing the promoter fusion element arealso established in such 96 well dishes. The test candidate substancesare added to the medium in individual wells containing both the test andcontrol cell lines set up in 96 well dishes. The cells are then brieflyexposed to UV or other stresses. 8 hr post stress induction, cells arewashed with PBS-A twice, and fixed with 0.5 ml containing 2% (v/v)formaldehyde and 0.2% glutaraldehyde in PBS for 5 min at roomtemperature. The cells are rinsed again with PBS and then stained with 2ml 5 mM potassium ferrocyanide, 5 mM potassium ferrocyanide, 2 mM MgCl₂and lmg/ml X-gal (diluted from a 40 mg/ml stock solution in dimethylsulfoxide) in PBS. Cells expressing β-Galactosidase were stained blueafter incubation at 37° C. for 23 days.

Results

The construct described above with the lacZ reporter gene was introducedinto PC12 cell line. A new cell line W5 was clonally established by G418selection. The W5 cell line was then tested for activation of the asequence promoter under suboptimal conditions names in 3 above. Theresults are: (a) The above cells, when exposed briefly to UV for 2minutes, turn blue upon staining the fixed cells with X-gal at 6-10 hrpost UV exposure. (b) The above cells, when exposed to HSV-1(F) virus atmultiplicity of infection of 5, also turn blue upon staining at 8 hrpost infection. (c) The above cells turn light blue when nerve growthfactor (rat, 7S) is introduced into the medium to allow differentiationprocesses. (d) The cells turn darker blue when Nerve Growth Factor isremoved from the medium after differentiation is complete and the cellshave become dependent on nerve growth factor for survival. (e) Little orno difference in color development is seen in cells starved for serum(0% fetal bovine serum) and those fully supplied in 10% fetal bovineserum. (f) The above experiments are repeated with control promoterfusion elements to control for the true inhibition of antiapoptosis geneexpression rather than toxicity-induced cell death. By this procedure,the positive candidates that can induce cell death in cells willtherefore render the following phenotypes: (i) Introduction of stress tothe test cell line in the absence of this substance will give rise toblue colored cells. (ii) Introduction of stress to the test cell line inthe presence of same substance will give rise to white cells. (iii)Introduction of stress to control cell lines with our without thisputative substance will have no effect on the color of cells.

The present invention has been disclosed in terms of specificembodiments which are believed by the inventors to be the best modes forcarrying out the invention. However, in light of the disclosure herebyprovided, those of skill in the various arts will recognize thatmodifications can be made without departing from the intended scope ofthe invention. The exemplary embodiments set forth herein and all othermodifications and embodiments are intended to be within the scope andspirit of the present invention and the appended claims.

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43 17 base pairs nucleic acid single linear DNA (genomic) unknown 1TTTAAAGTCG CGGCGGC 17 133 base pairs nucleic acid single linear DNA(genomic) unknown 2 GCAGCCCGGC CCCCCGCGGC CGAGACGAGC GAGTTAGACAGGCAAGCACT ACTCGCCTCT 60 GCACGCACAT GCTTGCCTGT CAAACTCTAC CACCCCGGCACGCTCTCTGT CTCCATGGCC 120 CGCCGCCGCC GCC 133 291 base pairs nucleic acidsingle linear DNA (genomic) unknown 3 ATCGCGGCCC CCGCCGCCCC CGGCCGCCCGGGCCCACGGG CGCCGTCCCA ACCGCACAGT 60 CCCAGGTAAC CTCCACGCCC AACTCGGAACCCGCGGTCAG GAGCGCGCCC GCGGCCGCCC 120 CGCCGCCGCC CCCCGCCAGT GGGCCCCCGCCTTCTTGTTC GCTGCTGCTG CGCCAGTGGC 180 TCCACGTTCC CGAGTCCGCG TCCGACGACGACGATGACGA CGACTGGCCG GACAGCCCCC 240 CGCCCGAGCC GGCGCCAGAG GCCCGGCCCACCGCCGCCGC CCCCCGCCCC C 291 595 base pairs nucleic acid single linearDNA (genomic) unknown 4 ACCGCCCGGC GCGGGCCCGG GGGGCGGGGC TAACCCCTCCCACCCCCCCT CACGCCCCTT 60 CCGCCTTCCG CCGCGCCTCG CCCTCCGCCT GCGCGTCACCGCAGAGCACC TGGCGCGCCT 120 GCGCCTGCGA CGCGCGGGCG GGGAGGGGGC GCCGGAGCCCCCCGCGACCC CCGCGACCCC 180 CGCGACCCCC GCGACCCCCG CGACCCCCGC GACCCCCGCGACCCCCGCGA CCCCCGCGAC 240 CCCCGCGACC CCCGCGCGGG TGCGCTTCTC GCCCCACGTCCGGGTGCGCC ACCTGGTGGT 300 CTGGGCCTCG GCCGCCCGCC TGGCGCGCCG CGGCTCGTGGGCCCGCGAGC GGGCCGACCG 360 GGCTCGGTTC CGGCGCCGGG TGGCGGAGGC CGAGGCGGTCATCGGGCCGT GCCTGGGGCC 420 CGAGGCCCGT GCCCGGGCCC TGGCCCGCGG AGCCGGCCCGGCGAACTCGG TCTAACGTTA 480 CACCCGAGGC GGCCTGGGTC TTCCGCGGAG CTCCCGGGAGCTCCGCACCA AGCCGCTCTC 540 CGGAGAGACG ATGGCAGGAG CCGCGCATAT ATACGCTGGGAGCCGGCCCG CCCCC 595 291 base pairs nucleic acid single linear DNA(genomic) unknown 5 GAGGCGGGCC CGCCCTCGGA GGGCGGGACT GGCCAATCGGCGGCCGCCAG CGCGGCGGGG 60 CCCGGCCAAC CAGCGTCCGC CGAGTCTTCG GGGCCCGGCCCACTGGGCGG GAGTTACCGC 120 CCAGTGGGCC GGGCCGCCCA CTTCCCGGTA TGGTAATTAAAAACTTACAA GAGGCCTTGT 180 TCCGCTTCCC GGTATGGTAA TTAGAAACTC ATTAATGGGCGGCCCCGGCC GCCCTTCCCG 240 CTTCCGGCAA TTCCCGCGGC CCTTAATGGG CAACCCCGGTATTCCCCGCC T 291 150 base pairs nucleic acid single linear DNA (genomic)unknown 6 TTTAAAGCGG TGGCGGCGGG CAGCCCGGGC CCCCCGCCGA GACTAGCGAGTTAGACAGGC 60 AAGCACTACT CGCCTCTGCA CGCACATGCT TGCCTGTCAA ACTCTACCACCCCGGCACGC 120 TCTCTGTCTC CATGGCCCGC CGCCGCCGCC 150 503 base pairsnucleic acid single linear DNA (genomic) unknown 7 ATCGCGGCCC CCGCCGCCCCCGGCCGCCCG GGCCCACGGG CGCCGTCCCA ACCGCACAGT 60 CCCAGGTAAC CTCCACGCCCAACTCGGAAC CCGCGGTCAG GAGCGCGCCC GCGGCCGCCC 120 CGCCGCCGCC CCCCGCCGGTGGGCCCCCGC CTTCTTGTTC GCTGCTGCTG CGCCAGTGGC 180 TCCACGTTCC CGAGTCCGCGTCCGACGACG ACGATGACGA CGACTGGCCG GACAGCCCCC 240 CGCCCGAGTC GGCGCCAGAGGCCCGGCCCA CCGCCGCCGC CCCCCGCCCC CCGGGCCCCC 300 ACCGCCCGGC GTGGGCCCGGGGGGCGGGGC TGACCCCTCC CACCCCCCCT CGCGCCCCTT 360 CCGCCTTCCG CCGCGCCTCGCCCTCCGCCT GCGCGTCACC GCGGAGCACC TGGCGCGCCT 420 GCGCCTGCGA CGCGCGGGCGGGGAGGGGGC GCCGGAGCCC CCCGCGACCC CCGCGACCCC 480 CGCGACCCCC GCGACCCCCGCGA 503 368 base pairs nucleic acid single linear DNA (genomic) unknown8 CCCCCGCGAC CCCCGCGCGG GTGCGCTTCT CGCCCCACGT CCGGGTGCGC CACCTGGTGG 60TCTGGGCCTC GGCCGCCCGC CTGGCGCGCC GCGGCTCGTG GGCCCGCGAG CGGGCCGACC 120GGGCTCGGTT CCGGCGCCGG GTGGCGGAGG CCGAGGCGGT CATCGGGCCG TGCCTGGGGC 180CCGAGGCCCG TGCCCGGGCC CTGGCCCGCG GAGCCGGCCC GGCGAACTCG GTCTAACGTT 240ACACCCGAGG CGGCCTGGGT CTTCCGCGGA GCTCCCGGGA GCTCCGCACC AAGCCGCTCT 300CCGGAGAGAC GATGGCAGGA GCCGCGCATA TATACGCTTG GAGCCAGCCC GCCCTCACAG 360GGCGGGCC 368 271 base pairs nucleic acid single linear DNA (genomic)unknown 9 GGGCGGGACT GGCCAATCGG CGGCCGCCAG CGCGGCGGGG CCCGGCCAACCAGCGTCCGC 60 CGAGTCTTCG GGGCCCGGCC CATTGGGCGG GAGTTACCGC CCAATGGGCCGGGCCGCCCA 120 CTTCCCGGTA TGGTAATTAA AAACTTGCAA GAGGCCTTGT TCCGCTTCCCGGTATGGTAA 180 TTAGAAACTC ATTAATGGGC GGCCCCGGCC GCCCTTCCCG CTTCCGGCAATTCCCGCGGC 240 CCTTAATGGG CAACCCCGGT ATTCCCCGCC T 271 11 base pairsnucleic acid single linear DNA (genomic) unknown 10 TTTAAAGTCA C 11 256base pairs nucleic acid single linear DNA (genomic) unknown 11AGCGGCGGGC AGCCCCCCCG CGGCCGAGAC TAGCGAGTTA GACAGGCAAG CACTACTCGC 60CTCTGCACGC ACATGCTTGC CTGTCAAACT CTACCACCCC GGCACGCTCT CTGTCTCCAT 120GGCCCGCCGC CGCCGCCGCC ATCGCGGCCC CCGCCGCCCC CGGCCGCCCG GGCCCACGGG 180CGCGGTCCCA ACCGCACAGT CCCAGGTAAC CTCCACGCCC AACTCGGAAC CCGTGGTCAG 240GAGCGCGCCC GCGGCC 256 154 base pairs nucleic acid single linear DNA(genomic) unknown 12 GGTGGGCCCC CGCCTTCTTG TTCGCTGCTG CTGCGCCAGTGGCTCCACGT TCCCGAGTCC 60 GCGTCCGACG ACGACGATGA CGACGACTGG CCGGACAGCCCCCCGCCCGA GCCGGCGCCA 120 GAGGCCCGGC CCACCGCCGC CGCCCCCCGC CCCC 154 212base pairs nucleic acid single linear DNA (genomic) unknown 13ACCGCCCGGC GCGGGCCCGG GGGGCGGGGC TAACCCCTCC CACCCCCCCT CACGCCCCTT 60CCGCCTTCCG CCGCGCCTCG CCCTCCGCCT GCGCGTCACC GCGGAGCACC TGGCGCGCCT 120GCGCCTGCGA CGCGCGGGCG GGGAGGGGGC GCCGAAGCCC CCCGCGACCC CCGCGACCCC 180CGCGACCCCC GCGACCCCCG CGACCCCCGC GA 212 356 base pairs nucleic acidsingle linear DNA (genomic) unknown 14 CCCCCGCGAC CCCCGCGCGG GTGCGCTTCTCGCCCCACGT CCGGGTGCGC CACCTGGTGG 60 TCTGGGCCTC GGCCGCCCGC CTGGCGCGCCGCGGCTCGTG GGCCCGCGAG CGGGCCGACC 120 GGGCTCGGTT CCGGCGCCGG GTGGCGGAGGCCGAGGCGGT CATCGGGCCG TGCCTGGGGC 180 CCGAGGCCCG TGCCCGGGCC CTGGCCCGCGGAGCCGGCCC GGCGAACTCG GTCTAACGTT 240 ACACCCGAGG CGGCCTGGGT CTTCCGCGGAGCTCCCGGGA GCTCCACACC AAGCCGCTCT 300 CCGGAGAGAC GATGGCAGGA GCCGCGCATATATACGCTGG GAGCCGGCCC GCCCCC 356 291 base pairs nucleic acid singlelinear DNA (genomic) unknown 15 GAGGCGGGCC CGCCCTCGGA GGGCGGGACTGGCCAATCGG CGGCCGCCAG CGCGGCGGGG 60 CCCGGCCAAC CAGCGTCCGC CGAGTCGTCGGGGCCCGGCC CACTGGGCGG TAACTCCCGC 120 CCAGTGGGCC GGGCCGCCCA CTTCCCGGTATGGTAATTAA AAACTTGCAA GAGGCCTTGT 180 TCCGCTTCCC GGTATGGTAA TTAGAAACTCATTAATGGGC GGCCCCGGCC GCCCTTCCCG 240 CTTCCGGCAA TTCCCGCGGC CCTTAATGGGCAACCCCGGT ATTCCCCGCC T 291 10 base pairs nucleic acid single linear DNA(genomic) unknown 16 TTTAAAGCGC 10 431 base pairs nucleic acid singlelinear DNA (genomic) unknown 17 GGCGGCGGGC AGCCCCCCCG CGGCCGAGACTAGCGAGTTA GACAGGCAAG CACTACTCGC 60 CTCTGCACGC ACATGCTTGC CTGTCAAACTCTACCACCCC GGCACGCTCT CTGTCTCCAT 120 GGCCCGCCGC CGCCGCCGCC ATCGCGGCCCCCGCCGCCCC CGGCCGCCCG GGCCCACGGG 180 CGCGGTCCCA ACCGCACAGT CCCAGGTAACCTCCACGCCC AACTCGGAAC CCGTGGTCAG 240 GAGCGCGCCC GCGGCCGCCC CGCCGCCGCCCCCCGCCGGT GGGCCCCCGC CTTCTTGTTC 300 GCTGCTGCTG CGGCAGTGGC TCCAGGTTCCGGAGTCCGCG TCCGACGACG ACGATGACGA 360 CGACTGGCCG GACAGCCCCC CGCCCGAGCCGGCGCCAGAG GCCCGGCCCA CCGCCGCCGC 420 CCCCCGCCCC C 431 212 base pairsnucleic acid single linear DNA (genomic) unknown 18 ACCGCCCGGCGCGGGCCCAG GGGGCGGGGC TGACCCCTCC CACCCCCCCT CACGCCCCTT 60 CCGCCTTCCGCCGCGCCTCG CCCTCCGCCT GCGCGTCACC GCAGAGCACC TGGCGCGCCT 120 GCGCCTGCGACGCGCGGGCG GGGAGGGGGC GCCGGAGCCC CCCGCGACCC CCGCGACCCC 180 CGCGACCCCCGCGACCCCCG CGACCCCCGC GA 212 356 base pairs nucleic acid single linearDNA (genomic) unknown 19 CCCCCGCGAC CCCCGCGCGG GTGCGCTTCT CGCCCCACGTCCGGGTGCGC CACCTGGTGG 60 TCTGGGCCTC GGCCGCCCGC CTGGCGCGCC GCGGCTCGTGGGCCCGCGAG CGGGCCGACC 120 GGGCTCGGTT CCGGCGCCGG GTGGCGGAGG CCGAGGCGGTCATCGGGCCG TGCCTGGGCC 180 CCAAGGCCCG CGCCCGGGCC CTGGCCCGCG GAGCCGGCCCGGCGAACTCG GTCTAACGTT 240 ACACCCGAGG CGGCCTGGGT CTTCCGCGGA GCTCCCGGGAGCTCCACACC AAGCCGCTCT 300 CCGGAGAGAC GATGGCAGGA GCCGCGCATA TATACGCTGGGAGCCGGCCC GCCCCC 356 291 base pairs nucleic acid single linear DNA(genomic) unknown 20 GAGGCGGGCC CGCCCTCGGA GGGCGGGACT GGCCAATCGGCGGCCGCCAG CGCGGCGGGG 60 CCCGGCCAAC CAGCGTCCGC CGAGTCGTCG GGGCCCGGCCCACTGGGCGG TAACTCCCGC 120 CCAGTGGGCC GGGCCGCCCA CTTCCCGGTA TGGTAATTAAAAACTTGCAA GAGGCCTTGT 180 TCCGCTTCCC GGTATGGTAA TTAGAAACTC ATTAATGGGCGGCCCCGGCC GCCCTTCCCG 240 CTTCCGGCAA TTCCCGCGGC CCTTAATGGG CAACCCCGGTATTCCCCGCC T 291 20 base pairs nucleic acid single linear DNA (genomic)unknown 21 GTAACCTAGA CTAGTCTAGC 20 20 base pairs nucleic acid singlelinear DNA (genomic) unknown 22 GTTACGCTAG ACTAGTCTAG 20 58 base pairsnucleic acid single linear DNA (genomic) unknown 23 CCCGGACATGGAACGAGTAC GACGACGCAG CCGACGCCGC CGGCGACCGG GCCCCGGG 58 51 base pairsnucleic acid single linear DNA (genomic) unknown 24 CTGCTCATGCTGCTGCGTCG GCTGCGGCGG CCGCTGGCCC GGGGCCCGTA C 51 6 amino acids aminoacid single linear peptide unknown 25 Met Ala Arg Arg Arg Arg 1 5 258amino acids amino acid single linear protein unknown 26 His Arg Gly ProArg Arg Pro Arg Pro Pro Gly Pro Thr Gly Ala Val 1 5 10 15 Pro Thr AlaGln Ser Gln Val Thr Ser Thr Pro Asn Ser Glu Pro Ala 20 25 30 Val Arg SerAla Pro Ala Ala Ala Pro Pro Pro Pro Pro Ala Ser Gly 35 40 45 Pro Pro ProSer Cys Ser Leu Leu Leu Arg Gln Trp Leu His Val Pro 50 55 60 Ala Glu SerAla Ser Asp Asp Asp Asp Asp Asp Asp Trp Pro Asp Ser 65 70 75 80 Pro ProPro Glu Pro Ala Pro Glu Ala Arg Pro Thr Ala Ala Ala Pro 85 90 95 Arg ProArg Ser Pro Pro Pro Gly Ala Gly Pro Gly Gly Gly Ala Asn 100 105 110 ProSer His Pro Pro Ser Arg Pro Phe Arg Leu Pro Pro Arg Leu Ala 115 120 125Leu Arg Leu Arg Val Thr Ala Glu His Leu Ala Arg Leu Arg Leu Arg 130 135140 Arg Ala Gly Gly Glu Gly Ala Pro Glu Pro Pro Ala Thr Pro Ala Thr 145150 155 160 Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala ThrPro 165 170 175 Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala Arg Val Arg PheSer Pro 180 185 190 His Val Arg Val Arg His Leu Val Val Trp Ala Ser AlaAla Arg Leu 195 200 205 Ala Arg Arg Gly Ser Trp Ala Arg Glu Arg Ala AspArg Ala Arg Phe 210 215 220 Arg Arg Arg Val Ala Glu Ala Glu Ala Val IleGly Pro Cys Leu Gly 225 230 235 240 Pro Glu Ala Arg Ala Arg Ala Leu AlaArg Gly Ala Gly Pro Ala Asn 245 250 255 Ser Val 6 amino acids amino acidsingle linear peptide unknown 27 Met Ala Arg Arg Arg Arg 1 5 169 aminoacids amino acid single linear protein unknown 28 His Arg Gly Pro ArgArg Pro Arg Pro Pro Gly Pro Thr Gly Ala Val 1 5 10 15 Pro Thr Ala GlnSer Gln Val Thr Ser Thr Pro Asn Ser Glu Pro Ala 20 25 30 Val Arg Ser AlaPro Ala Ala Ala Pro Pro Pro Pro Pro Ala Gly Gly 35 40 45 Pro Pro Pro SerCys Ser Leu Leu Leu Arg Gln Trp Leu His Val Pro 50 55 60 Glu Ser Ala SerAsp Asp Asp Asp Asp Asp Asp Trp Pro Asp Ser Pro 65 70 75 80 Pro Pro GluSer Ala Pro Glu Ala Arg Pro Thr Ala Ala Ala Pro Arg 85 90 95 Pro Pro GlyPro His Arg Pro Ala Trp Ala Arg Gly Ala Gly Leu Thr 100 105 110 Pro ProThr Pro Pro Arg Ala Pro Ser Ala Phe Arg Arg Ala Ser Pro 115 120 125 SerAla Cys Ala Ser Pro Arg Ser Thr Trp Arg Ala Cys Ala Cys Asp 130 135 140Ala Arg Ala Gly Arg Gly Arg Arg Ser Pro Pro Arg Pro Pro Arg Pro 145 150155 160 Pro Arg Pro Pro Arg Pro Pro Arg Pro 165 180 amino acids aminoacid single linear protein unknown 29 Pro Arg Gly Cys Ala Ser Arg ProThr Ser Gly Cys Ala Thr Trp Trp 1 5 10 15 Ser Gly Pro Arg Pro Pro AlaTrp Arg Ala Ala Ala Arg Gly Pro Ala 20 25 30 Ser Gly Pro Thr Gly Leu GlySer Gly Ala Gly Trp Arg Arg Pro Arg 35 40 45 Arg Ser Ser Gly Arg Ala TrpGly Pro Arg Pro Val Pro Gly Pro Trp 50 55 60 Pro Ala Glu Pro Ala Arg ArgThr Arg Ser Asn Val Thr Pro Glu Ala 65 70 75 80 Ala Trp Val Phe Arg GlyAla Pro Gly Ser Ser Ala Pro Ser Arg Ser 85 90 95 Pro Glu Arg Arg Trp GlnGlu Pro Arg Ile Tyr Thr Leu Gly Ala Ser 100 105 110 Pro Pro Ser Gln GlyGly Pro Pro Arg Gly Arg Asp Trp Pro Ile Gly 115 120 125 Gly Arg Gln ArgGly Gly Ala Arg Pro Thr Ser Val Arg Arg Val Phe 130 135 140 Gly Ala ArgPro Ile Gly Arg Glu Leu Pro Pro Asn Gly Pro Gly Arg 145 150 155 160 ProLeu Pro Gly Met Val Ile Lys Asn Leu Gln Glu Ala Leu Phe Arg 165 170 175Phe Pro Val Trp 180 46 amino acids amino acid single linear proteinunknown 30 Met Ala Arg Arg Arg Arg Arg His Arg Gly Pro Arg Arg Pro ArgPro 1 5 10 15 Pro Gly Pro Thr Gly Ala Val Pro Thr Ala Gln Ser Gln ValThr Ser 20 25 30 Thr Pro Asn Ser Glu Pro Val Val Arg Ser Ala Pro Ala Ala35 40 45 126 amino acids amino acid single linear protein unknown 31 GlyGly Pro Pro Pro Ser Cys Ser Leu Leu Leu Arg Gln Trp Leu His 1 5 10 15Val Pro Glu Ser Ala Ser Asp Asp Asp Asp Asp Asp Asp Trp Pro Asp 20 25 30Ser Pro Pro Pro Glu Pro Ala Pro Glu Ala Arg Pro Thr Ala Ala Ala 35 40 45Pro Arg Pro Arg Ser Pro Pro Pro Gly Ala Gly Pro Gly Gly Gly Ala 50 55 60Asn Pro Ser His Pro Pro Ser Arg Pro Phe Arg Leu Pro Pro Arg Leu 65 70 7580 Ala Leu Arg Leu Arg Val Thr Ala Glu His Leu Ala Arg Leu Arg Leu 85 9095 Arg Arg Ala Gly Gly Glu Gly Ala Pro Lys Pro Pro Ala Thr Pro Ala 100105 110 Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro 115 120125 73 amino acids amino acid single linear protein unknown 32 Ala ArgVal Arg Phe Ser Pro His Val Arg Val Arg His Leu Val Val 1 5 10 15 TrpAla Ser Ala Ala Arg Leu Ala Arg Arg Gly Ser Trp Ala Arg Glu 20 25 30 ArgAla Asp Arg Ala Arg Phe Arg Arg Arg Val Ala Glu Ala Glu Ala 35 40 45 ValIle Gly Pro Cys Leu Gly Pro Glu Ala Arg Ala Arg Ala Leu Ala 50 55 60 ArgGly Ala Gly Pro Ala Asn Ser Val 65 70 179 amino acids amino acid singlelinear protein unknown 33 Met Ala Arg Arg Arg Arg Arg His Arg Gly ProArg Arg Pro Arg Pro 1 5 10 15 Pro Gly Pro Thr Gly Ala Val Pro Thr AlaGln Ser Gln Val Thr Ser 20 25 30 Thr Pro Asn Ser Glu Pro Val Val Arg SerAla Pro Ala Ala Ala Pro 35 40 45 Pro Pro Pro Pro Ala Gly Gly Pro Pro ProSer Cys Ser Leu Leu Leu 50 55 60 Arg Gln Trp Leu Gln Val Pro Glu Ser AlaSer Asp Asp Asp Asp Asp 65 70 75 80 Asp Asp Trp Pro Asp Ser Pro Pro ProGlu Pro Ala Pro Glu Ala Arg 85 90 95 Pro Thr Ala Ala Ala Pro Arg Pro ArgSer Pro Pro Pro Gly Ala Gly 100 105 110 Pro Gly Gly Gly Ala Asp Pro SerHis Pro Pro Ser Arg Pro Phe Arg 115 120 125 Leu Pro Pro Arg Leu Ala LeuArg Leu Arg Val Thr Ala Glu His Leu 130 135 140 Ala Arg Leu Arg Leu ArgArg Ala Gly Gly Glu Gly Ala Pro Glu Pro 145 150 155 160 Pro Ala Thr ProAla Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro 165 170 175 Ala Thr Pro73 amino acids amino acid single linear protein unknown 34 Ala Arg ValArg Phe Ser Pro His Val Arg Val Arg His Leu Val Val 1 5 10 15 Trp AlaSer Ala Ala Arg Leu Ala Arg Arg Gly Ser Trp Ala Arg Glu 20 25 30 Arg AlaAsp Arg Ala Arg Phe Arg Arg Arg Val Ala Glu Ala Glu Ala 35 40 45 Val IleGly Pro Cys Leu Gly Lys Glu Ala Arg Ala Arg Ala Leu Ala 50 55 60 Arg GlyAla Gly Pro Ala Asn Ser Val 65 70 18 amino acids amino acid singlelinear peptide unknown 35 Met Asp Glu Tyr Asp Asp Ala Ala Asp Ala AlaGly Asp Arg Ala Pro 1 5 10 15 Gly Met 1327 base pairs nucleic acidsingle linear DNA (genomic) unknown 36 TTTAAAGTCG CGGCGGCGCA GCCCGGCCCCCCGCGGCCGA GACGAGCGAG TTAGACAGGC 60 AAGCACTACT CGCCTCTGCA CGCACATGCTTGCCTGTCAA ACTCTACCAC CCCGGCACGC 120 TCTCTGTCTC CATGGCCCGC CGCCGCCGCCATCGCGGCCC CCGCCGCCCC CGGCCGCCCG 180 GGCCCACGGG CGCCGTCCCA ACCGCACAGTCCCAGGTAAC CTCCACGCCC AACTCGGAAC 240 CCGCGGTCAG GAGCGCGCCC GCGGCCGCCCCGCCGCCGCC CCCCGCCAGT GGGCCCCCGC 300 CTTCTTGTTC GCTGCTGCTG CGCCAGTGGCTCCACGTTCC CGAGTCCGCG TCCGACGACG 360 ACGATGACGA CGACTGGCCG GACAGCCCCCCGCCCGAGCC GGCGCCAGAG GCCCGGCCCA 420 CCGCCGCCGC CCCCCGCCCC CACCGCCCGGCGCGGGCCCG GGGGGCGGGG CTAACCCCTC 480 CCACCCCCCC TCACGCCCCT TCCGCCTTCCGCCGCGCCTC GCCCTCCGCC TGCGCGTCAC 540 CGCAGAGCAC CTGGCGCGCC TGCGCCTGCGACGCGCGGGC GGGGAGGGGG CGCCGGAGCC 600 CCCCGCGACC CCCGCGACCC CCGCGACCCCCGCGACCCCC GCGACCCCCG CGACCCCCGC 660 GACCCCCGCG ACCCCCGCGA CCCCCGCGACCCCCGCGCGG GTGCGCTTCT CGCCCCACGT 720 CCGGGTGCGC CACCTGGTGG TCTGGGCCTCGGCCGCCCGC CTGGCGCGCC GCGGCTCGTG 780 GGCCCGCGAG CGGGCCGACC GGGCTCGGTTCCGGCGCCGG GTGGCGGAGG CCGAGGCGGT 840 CATCGGGCCG TGCCTGGGGC CCGAGGCCCGTGCCCGGGCC CTGGCCCGCG GAGCCGGCCC 900 GGCGAACTCG GTCTAACGTT ACACCCGAGGCGGCCTGGGT CTTCCGCGGA GCTCCCGGGA 960 GCTCCGCACC AAGCCGCTCT CCGGAGAGACGATGGCAGGA GCCGCGCATA TATACGCTGG 1020 GAGCCGGCCC GCCCCCGAGG CGGGCCCGCCCTCGGAGGGC GGGACTGGCC AATCGGCGGC 1080 CGCCAGCGCG GCGGGGCCCG GCCAACCAGCGTCCGCCGAG TCTTCGGGGC CCGGCCCACT 1140 GGGCGGGAGT TACCGCCCAG TGGGCCGGGCCGCCCACTTC CCGGTATGGT AATTAAAAAC 1200 TTACAAGAGG CCTTGTTCCG CTTCCCGGTATGGTAATTAG AAACTCATTA ATGGGCGGCC 1260 CCGGCCGCCC TTCCCGCTTC CGGCAATTCCCGCGGCCCTT AATGGGCAAC CCCGGTATTC 1320 CCCGCCT 1327 1292 base pairsnucleic acid single linear DNA (genomic) unknown 37 TTTAAAGCGGTGGCGGCGGG CAGCCCGGGC CCCCCGCCGA GACTAGCGAG TTAGACAGGC 60 AAGCACTACTCGCCTCTGCA CGCACATGCT TGCCTGTCAA ACTCTACCAC CCCGGCACGC 120 TCTCTGTCTCCATGGCCCGC CGCCGCCGCC ATCGCGGCCC CCGCCGCCCC CGGCCGCCCG 180 GGCCCACGGGCGCCGTCCCA ACCGCACAGT CCCAGGTAAC CTCCACGCCC AACTCGGAAC 240 CCGCGGTCAGGAGCGCGCCC GCGGCCGCCC CGCCGCCGCC CCCCGCCGGT GGGCCCCCGC 300 CTTCTTGTTCGCTGCTGCTG CGCCAGTGGC TCCACGTTCC CGAGTCCGCG TCCGACGACG 360 ACGATGACGACGACTGGCCG GACAGCCCCC CGCCCGAGTC GGCGCCAGAG GCCCGGCCCA 420 CCGCCGCCGCCCCCCGCCCC CCGGGCCCCC ACCGCCCGGC GTGGGCCCGG GGGGCGGGGC 480 TGACCCCTCCCACCCCCCCT CGCGCCCCTT CCGCCTTCCG CCGCGCCTCG CCCTCCGCCT 540 GCGCGTCACCGCGGAGCACC TGGCGCGCCT GCGCCTGCGA CGCGCGGGCG GGGAGGGGGC 600 GCCGGAGCCCCCCGCGACCC CCGCGACCCC CGCGACCCCC GCGACCCCCG CGACCCCCGC 660 GACCCCCGCGCGGGTGCGCT TCTCGCCCCA CGTCCGGGTG CGCCACCTGG TGGTCTGGGC 720 CTCGGCCGCCCGCCTGGCGC GCCGCGGCTC GTGGGCCCGC GAGCGGGCCG ACCGGGCTCG 780 GTTCCGGCGCCGGGTGGCGG AGGCCGAGGC GGTCATCGGG CCGTGCCTGG GGCCCGAGGC 840 CCGTGCCCGGGCCCTGGCCC GCGGAGCCGG CCCGGCGAAC TCGGTCTAAC GTTACACCCG 900 AGGCGGCCTGGGTCTTCCGC GGAGCTCCCG GGAGCTCCGC ACCAAGCCGC TCTCCGGAGA 960 GACGATGGCAGGAGCCGCGC ATATATACGC TTGGAGCCAG CCCGCCCTCA CAGGGCGGGC 1020 CGGGCGGGACTGGCCAATCG GCGGCCGCCA GCGCGGCGGG GCCCGGCCAA CCAGCGTCCG 1080 CCGAGTCTTCGGGGCCCGGC CCATTGGGCG GGAGTTACCG CCCAATGGGC CGGGCCGCCC 1140 ACTTCCCGGTATGGTAATTA AAAACTTGCA AGAGGCCTTG TTCCGCTTCC CGGTATGGTA 1200 ATTAGAAACTCATTAATGGG CGGCCCCGGC CGCCCTTCCC GCTTCCGGCA ATTCCCGCGG 1260 CCCTTAATGGGCAACCCCGG TATTCCCCGC CT 1292 1280 base pairs nucleic acid single linearDNA (genomic) unknown 38 TTTAAAGTCA CAGCGGCGGG CAGCCCCCCC GCGGCCGAGACTAGCGAGTT AGACAGGCAA 60 GCACTACTCG CCTCTGCACG CACATGCTTG CCTGTCAAACTCTACCACCC CGGCACGCTC 120 TCTGTCTCCA TGGCCCGCCG CCGCCGCCGC CATCGCGGCCCCCGCCGCCC CCGGCCGCCC 180 GGGCCCACGG GCGCGGTCCC AACCGCACAG TCCCAGGTAACCTCCACGCC CAACTCGGAA 240 CCCGTGGTCA GGAGCGCGCC CGCGGCCGGT GGGCCCCCGCCTTCTTGTTC GCTGCTGCTG 300 CGCCAGTGGC TCCACGTTCC CGAGTCCGCG TCCGACGACGACGATGACGA CGACTGGCCG 360 GACAGCCCCC CGCCCGAGCC GGCGCCAGAG GCCCGGCCCACCGCCGCCGC CCCCCGCCCC 420 CACCGCCCGG CGCGGGCCCG GGGGGCGGGG CTAACCCCTCCCACCCCCCC TCACGCCCCT 480 TCCGCCTTCC GCCGCGCCTC GCCCTCCGCC TGCGCGTCACCGCGGAGCAC CTGGCGCGCC 540 TGCGCCTGCG ACGCGCGGGC GGGGAGGGGG CGCCGAAGCCCCCCGCGACC CCCGCGACCC 600 CCGCGACCCC CGCGACCCCC GCGACCCCCG CGACCCCCGCGACCCCCGCG CGGGTGCGCT 660 TCTCGCCCCA CGTCCGGGTG CGCCACCTGG TGGTCTGGGCCTCGGCCGCC CGCCTGGCGC 720 GCCGCGGCTC GTGGGCCCGC GAGCGGGCCG ACCGGGCTCGGTTCCGGCGC CGGGTGGCGG 780 AGGCCGAGGC GGTCATCGGG CCGTGCCTGG GGCCCGAGGCCCGTGCCCGG GCCCTGGCCC 840 GCGGAGCCGG CCCGGCGAAC TCGGTCTAAC GTTACACCCGAGGCGGCCTG GGTCTTCCGC 900 GGAGCTCCCG GGAGCTCCAC ACCAAGCCGC TCTCCGGAGAGACGATGGCA GGAGCCGCGC 960 ATATATACGC TGGGAGCCGG CCCGCCCCCG AGGCGGGCCCGCCCTCGGAG GGCGGGACTG 1020 GCCAATCGGC GGCCGCCAGC GCGGCGGGGC CCGGCCAACCAGCGTCCGCC GAGTCGTCGG 1080 GGCCCGGCCC ACTGGGCGGT AACTCCCGCC CAGTGGGCCGGGCCGCCCAC TTCCCGGTAT 1140 GGTAATTAAA AACTTGCAAG AGGCCTTGTT CCGCTTCCCGGTATGGTAAT TAGAAACTCA 1200 TTAATGGGCG GCCCCGGCCG CCCTTCCCGC TTCCGGCAATTCCCGCGGCC CTTAATGGGC 1260 AACCCCGGTA TTCCCCGCCT 1280 1300 base pairsnucleic acid single linear DNA (genomic) unknown 39 TTTAAAGCGCGGCGGCGGGC AGCCCCCCCG CGGCCGAGAC TAGCGAGTTA GACAGGCAAG 60 CACTACTCGCCTCTGCACGC ACATGCTTGC CTGTCAAACT CTACCACCCC GGCACGCTCT 120 CTGTCTCCATGGCCCGCCGC CGCCGCCGCC ATCGCGGCCC CCGCCGCCCC CGGCCGCCCG 180 GGCCCACGGGCGCGGTCCCA ACCGCACAGT CCCAGGTAAC CTCCACGCCC AACTCGGAAC 240 CCGTGGTCAGGAGCGCGCCC GCGGCCGCCC CGCCGCCGCC CCCCGCCGGT GGGCCCCCGC 300 CTTCTTGTTCGCTGCTGCTG CGGCAGTGGC TCCAGGTTCC GGAGTCCGCG TCCGACGACG 360 ACGATGACGACGACTGGCCG GACAGCCCCC CGCCCGAGCC GGCGCCAGAG GCCCGGCCCA 420 CCGCCGCCGCCCCCCGCCCC CACCGCCCGG CGCGGGCCCA GGGGGCGGGG CTGACCCCTC 480 CCACCCCCCCTCACGCCCCT TCCGCCTTCC GCCGCGCCTC GCCCTCCGCC TGCGCGTCAC 540 CGCAGAGCACCTGGCGCGCC TGCGCCTGCG ACGCGCGGGC GGGGAGGGGG CGCCGGAGCC 600 CCCCGCGACCCCCGCGACCC CCGCGACCCC CGCGACCCCC GCGACCCCCG CGACCCCCGC 660 GACCCCCGCGCGGGTGCGCT TCTCGCCCCA CGTCCGGGTG CGCCACCTGG TGGTCTGGGC 720 CTCGGCCGCCCGCCTGGCGC GCCGCGGCTC GTGGGCCCGC GAGCGGGCCG ACCGGGCTCG 780 GTTCCGGCGCCGGGTGGCGG AGGCCGAGGC GGTCATCGGG CCGTGCCTGG GCCCCAAGGC 840 CCGCGCCCGGGCCCTGGCCC GCGGAGCCGG CCCGGCGAAC TCGGTCTAAC GTTACACCCG 900 AGGCGGCCTGGGTCTTCCGC GGAGCTCCCG GGAGCTCCAC ACCAAGCCGC TCTCCGGAGA 960 GACGATGGCAGGAGCCGCGC ATATATACGC TGGGAGCCGG CCCGCCCCCG AGGCGGGCCC 1020 GCCCTCGGAGGGCGGGACTG GCCAATCGGC GGCCGCCAGC GCGGCGGGGC CCGGCCAACC 1080 AGCGTCCGCCGAGTCGTCGG GGCCCGGCCC ACTGGGCGGT AACTCCCGCC CAGTGGGCCG 1140 GGCCGCCCACTTCCCGGTAT GGTAATTAAA AACTTGCAAG AGGCCTTGTT CCGCTTCCCG 1200 GTATGGTAATTAGAAACTCA TTAATGGGCG GCCCCGGCCG CCCTTCCCGC TTCCGGCAAT 1260 TCCCGCGGCCCTTAATGGGC AACCCCGGTA TTCCCCGCCT 1300 264 amino acids amino acid singlelinear protein unknown 40 Met Ala Arg Arg Arg Arg His Arg Gly Pro ArgArg Pro Arg Pro Pro 1 5 10 15 Gly Pro Thr Gly Ala Val Pro Thr Ala GlnSer Gln Val Thr Ser Thr 20 25 30 Pro Asn Ser Glu Pro Ala Val Arg Ser AlaPro Ala Ala Ala Pro Pro 35 40 45 Pro Pro Pro Ala Ser Gly Pro Pro Pro SerCys Ser Leu Leu Leu Arg 50 55 60 Gln Trp Leu His Val Pro Ala Glu Ser AlaSer Asp Asp Asp Asp Asp 65 70 75 80 Asp Asp Trp Pro Asp Ser Pro Pro ProGlu Pro Ala Pro Glu Ala Arg 85 90 95 Pro Thr Ala Ala Ala Pro Arg Pro ArgSer Pro Pro Pro Gly Ala Gly 100 105 110 Pro Gly Gly Gly Ala Asn Pro SerHis Pro Pro Ser Arg Pro Phe Arg 115 120 125 Leu Pro Pro Arg Leu Ala LeuArg Leu Arg Val Thr Ala Glu His Leu 130 135 140 Ala Arg Leu Arg Leu ArgArg Ala Gly Gly Glu Gly Ala Pro Glu Pro 145 150 155 160 Pro Ala Thr ProAla Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro 165 170 175 Ala Thr ProAla Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala 180 185 190 Arg ValArg Phe Ser Pro His Val Arg Val Arg His Leu Val Val Trp 195 200 205 AlaSer Ala Ala Arg Leu Ala Arg Arg Gly Ser Trp Ala Arg Glu Arg 210 215 220Ala Asp Arg Ala Arg Phe Arg Arg Arg Val Ala Glu Ala Glu Ala Val 225 230235 240 Ile Gly Pro Cys Leu Gly Pro Glu Ala Arg Ala Arg Ala Leu Ala Arg245 250 255 Gly Ala Gly Pro Ala Asn Ser Val 260 355 amino acids aminoacid single linear protein unknown 41 Met Ala Arg Arg Arg Arg His ArgGly Pro Arg Arg Pro Arg Pro Pro 1 5 10 15 Gly Pro Thr Gly Ala Val ProThr Ala Gln Ser Gln Val Thr Ser Thr 20 25 30 Pro Asn Ser Glu Pro Ala ValArg Ser Ala Pro Ala Ala Ala Pro Pro 35 40 45 Pro Pro Pro Ala Gly Gly ProPro Pro Ser Cys Ser Leu Leu Leu Arg 50 55 60 Gln Trp Leu His Val Pro GluSer Ala Ser Asp Asp Asp Asp Asp Asp 65 70 75 80 Asp Trp Pro Asp Ser ProPro Pro Glu Ser Ala Pro Glu Ala Arg Pro 85 90 95 Thr Ala Ala Ala Pro ArgPro Pro Gly Pro His Arg Pro Ala Trp Ala 100 105 110 Arg Gly Ala Gly LeuThr Pro Pro Thr Pro Pro Arg Ala Pro Ser Ala 115 120 125 Phe Arg Arg AlaSer Pro Ser Ala Cys Ala Ser Pro Arg Ser Thr Trp 130 135 140 Arg Ala CysAla Cys Asp Ala Arg Ala Gly Arg Gly Arg Arg Ser Pro 145 150 155 160 ProArg Pro Pro Arg Pro Pro Arg Pro Pro Arg Pro Pro Arg Pro Pro 165 170 175Arg Gly Cys Ala Ser Arg Pro Thr Ser Gly Cys Ala Thr Trp Trp Ser 180 185190 Gly Pro Arg Pro Pro Ala Trp Arg Ala Ala Ala Arg Gly Pro Ala Ser 195200 205 Gly Pro Thr Gly Leu Gly Ser Gly Ala Gly Trp Arg Arg Pro Arg Arg210 215 220 Ser Ser Gly Arg Ala Trp Gly Pro Arg Pro Val Pro Gly Pro TrpPro 225 230 235 240 Ala Glu Pro Ala Arg Arg Thr Arg Ser Asn Val Thr ProGlu Ala Ala 245 250 255 Trp Val Phe Arg Gly Ala Pro Gly Ser Ser Ala ProSer Arg Ser Pro 260 265 270 Glu Arg Arg Trp Gln Glu Pro Arg Ile Tyr ThrLeu Gly Ala Ser Pro 275 280 285 Pro Ser Gln Gly Gly Pro Pro Arg Gly ArgAsp Trp Pro Ile Gly Gly 290 295 300 Arg Gln Arg Gly Gly Ala Arg Pro ThrSer Val Arg Arg Val Phe Gly 305 310 315 320 Ala Arg Pro Ile Gly Arg GluLeu Pro Pro Asn Gly Pro Gly Arg Pro 325 330 335 Leu Pro Gly Met Val IleLys Asn Leu Gln Glu Ala Leu Phe Arg Phe 340 345 350 Pro Val Trp 355 245amino acids amino acid single linear protein unknown 42 Met Ala Arg ArgArg Arg Arg His Arg Gly Pro Arg Arg Pro Arg Pro 1 5 10 15 Pro Gly ProThr Gly Ala Val Pro Thr Ala Gln Ser Gln Val Thr Ser 20 25 30 Thr Pro AsnSer Glu Pro Val Val Arg Ser Ala Pro Ala Ala Gly Gly 35 40 45 Pro Pro ProSer Cys Ser Leu Leu Leu Arg Gln Trp Leu His Val Pro 50 55 60 Glu Ser AlaSer Asp Asp Asp Asp Asp Asp Asp Trp Pro Asp Ser Pro 65 70 75 80 Pro ProGlu Pro Ala Pro Glu Ala Arg Pro Thr Ala Ala Ala Pro Arg 85 90 95 Pro ArgSer Pro Pro Pro Gly Ala Gly Pro Gly Gly Gly Ala Asn Pro 100 105 110 SerHis Pro Pro Ser Arg Pro Phe Arg Leu Pro Pro Arg Leu Ala Leu 115 120 125Arg Leu Arg Val Thr Ala Glu His Leu Ala Arg Leu Arg Leu Arg Arg 130 135140 Ala Gly Gly Glu Gly Ala Pro Lys Pro Pro Ala Thr Pro Ala Thr Pro 145150 155 160 Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala Arg ValArg 165 170 175 Phe Ser Pro His Val Arg Val Arg His Leu Val Val Trp AlaSer Ala 180 185 190 Ala Arg Leu Ala Arg Arg Gly Ser Trp Ala Arg Glu ArgAla Asp Arg 195 200 205 Ala Arg Phe Arg Arg Arg Val Ala Glu Ala Glu AlaVal Ile Gly Pro 210 215 220 Cys Leu Gly Pro Glu Ala Arg Ala Arg Ala LeuAla Arg Gly Ala Gly 225 230 235 240 Pro Ala Asn Ser Val 245 252 aminoacids amino acid single linear protein unknown 43 Met Ala Arg Arg ArgArg Arg His Arg Gly Pro Arg Arg Pro Arg Pro 1 5 10 15 Pro Gly Pro ThrGly Ala Val Pro Thr Ala Gln Ser Gln Val Thr Ser 20 25 30 Thr Pro Asn SerGlu Pro Val Val Arg Ser Ala Pro Ala Ala Ala Pro 35 40 45 Pro Pro Pro ProAla Gly Gly Pro Pro Pro Ser Cys Ser Leu Leu Leu 50 55 60 Arg Gln Trp LeuGln Val Pro Glu Ser Ala Ser Asp Asp Asp Asp Asp 65 70 75 80 Asp Asp TrpPro Asp Ser Pro Pro Pro Glu Pro Ala Pro Glu Ala Arg 85 90 95 Pro Thr AlaAla Ala Pro Arg Pro Arg Ser Pro Pro Pro Gly Ala Gly 100 105 110 Pro GlyGly Gly Ala Asp Pro Ser His Pro Pro Ser Arg Pro Phe Arg 115 120 125 LeuPro Pro Arg Leu Ala Leu Arg Leu Arg Val Thr Ala Glu His Leu 130 135 140Ala Arg Leu Arg Leu Arg Arg Ala Gly Gly Glu Gly Ala Pro Glu Pro 145 150155 160 Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro165 170 175 Ala Thr Pro Ala Arg Val Arg Phe Ser Pro His Val Arg Val ArgHis 180 185 190 Leu Val Val Trp Ala Ser Ala Ala Arg Leu Ala Arg Arg GlySer Trp 195 200 205 Ala Arg Glu Arg Ala Asp Arg Ala Arg Phe Arg Arg ArgVal Ala Glu 210 215 220 Ala Glu Ala Val Ile Gly Pro Cys Leu Gly Lys GluAla Arg Ala Arg 225 230 235 240 Ala Leu Ala Arg Gly Ala Gly Pro Ala AsnSer Val 245 250

What is claimed is:
 1. A method of treating tumorigenic disease of thecentral nervous system in a mammal comprising the step of administeringat or near a site of a tumor of said tumorineic disease a herpes simplexvirus vector lacking expressible γ₁34.5 genes.
 2. The method of claim 1wherein the γ₁34.5 gene comprises a stop codon in reading frame.
 3. Themethod of claim 1 wherein the γ₁34.5 gene comprises a deletion mutation.4. The method of claim 1 wherein the herpes simplex virus vector isHSV-1.
 5. The method of claim 1 wherein the herpes simplex virus vectoris HSV-2.
 6. The method of claim 1 wherein the herpes simplex virusvector is administered via direct injection into said tumor of saidtumorigenic disease.